ROR1 (NTRKR1) specific chimeric antigen receptors for cancer immunotherapy

ABSTRACT

The present invention relates to Chimeric Antigen Receptors (CAR) that are recombinant chimeric proteins able to redirect immune cell specificity and reactivity toward selected membrane antigens, and more particularly in which extracellular ligand binding is a scFV derived from a ROR1 monoclonal antibody, conferring specific immunity against ROR1 positive cells. The engineered immune cells endowed with such CARs are particularly suited for treating lymphomas and leukemia, and for solid tumors such as breast, colon, lung, and kidney tumors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Stage entry of International Application No. PCT/EP2015/067444, filed Jul. 29, 2015, which claims priority to Danish Patent Application No. PA201470465, filed Jul. 29, 2014. The disclosure of the prior applications are hereby incorporated in their entirety by reference.

FIELD OF THE INVENTION

The present invention relates to Chimeric Antigen Receptors (CAR) that are recombinant chimeric proteins able to redirect immune cell specificity and reactivity toward ROR1, a cell surface glycoprotein found on most myeloid cells and used to diagnose chronic lymphocytic leukemia (CLL) or solid tumors such as breast, colon, lung, and kidney tumors in patients. The CARs according to the invention are particularly useful to treat malignant cells bearing ROR1 antigen, when expressed in T-cells or NK cells. The resulting engineered immune cells display high level of specificity toward malignant cells, conferring safety and efficiency for immunotherapy.

BACKGROUND OF THE INVENTION

Adoptive immunotherapy, which involves the transfer of autologous antigen-specific T cells generated ex vivo, is a promising strategy to treat viral infections and cancer. The T cells used for adoptive immunotherapy can be generated either by expansion of antigen-specific T cells or redirection of T cells through genetic engineering (Park, Rosenberg et al. 2011). Transfer of viral antigen specific T cells is a well-established procedure used for the treatment of transplant associated viral infections and rare viral-related malignancies. Similarly, isolation and transfer of tumor specific T cells has been shown to be successful in treating melanoma.

Novel specificities in T cells have been successfully generated through the genetic transfer of transgenic T cell receptors or chimeric antigen receptors (CARs) (Jena, Dotti et al. 2010). A schematic representation for the 3 generations of CARs is presented in FIG. 1. CARs are synthetic receptors consisting of a targeting moiety that is associated with one or more signaling domains in a single fusion molecule. In general, the binding moiety of a CAR consists of an antigen-binding domain of a single-chain antibody (scFv), comprising the light and variable fragments of a monoclonal antibody joined by a flexible linker. Binding moieties based on receptor or ligand domains have also been used successfully. The signaling domains for first generation CARs are derived from the cytoplasmic region of the CD3zeta or the Fc receptor gamma chains. First generation CARs have been shown to successfully redirect T-cell cytotoxicity. However, they failed to provide prolonged expansion and anti-tumor activity in vivo. Signaling domains from co-stimulatory molecules, as well as transmembrane and hinge domains have been added to form CARs of second and third generations, leading to some successful therapeutic trials in humans, where T-cells could be redirected against malignant cells expressing CD19 (June et al., 2011). However, the particular combination of signaling domains, transmembrane and co-stimulatory domains used with respect to CD19 ScFv, was rather antigen-specific and cannot be expanded to any antigen markers.

Chronic lymphocytic leukemia (CLL) is one of the most commonly diagnosed leukemias managed by practicing hematologists. For many years patients with CLL have been viewed as similar, with a long natural history and only marginally effective therapies that rarely yielded complete responses. Recently, several important observations related to the biologic significance of V_(H) mutational status and associated ZAP-70 overexpression, disrupted p53 function, and chromosomal aberrations have led to the ability to identify patients at high risk for early disease progression and inferior survival. Concurrent with these investigations, several treatments including the nucleoside analogues, monoclonal antibodies rituximab and alemtuzumab have been introduced. Combination of these therapies in clinical trials has led to high complete and overall response rates when applied as initial therapy for symptomatic CLL. Thus, the complexity of initial risk stratification of CLL and treatment has increased significantly. Furthermore, when these initial therapies do not work, approach of the CLL patient with fludarabine-refractory disease can be quite challenging (Byrd J. C et al, 2014).

One candidate antigen of immunotherapies for chronic lymphocytic leukemia (CLL) is Tyrosine-protein kinase transmembrane receptor ROR1 (also called NTRKR1; UniProtKB/TrEMBL) entries: Q01973). ROR1 (The receptor tyrosine kinase-like orphan receptor 1) is a 120-kDa glycoprotein containing an extracellular immunoglobulin (Ig)-like, Kringle, and Frizzled-like cysteine rich domain (FIG. 2). The protein encoded by this gene is a receptor tyrosine kinase that modulates neurite growth in the central nervous system. It is a type I membrane protein and belongs to the ROR subfamily of cell surface receptors (Reddy et al, 1997). Although ROR1 protein expression in patients with CLL with respect to normal leukocytes and its role in the pathobiology of CLL merits further studies. ROR1 may be an appropriate target for cancer immunotherapy (Daneshmanesh et al; 2008). ROR1 is indeed expressed on a variety of B-cell malignancies, but also on subsets of some solid tumors, including breast, colon, lung, and kidney tumors. It is believed that ROR1 functions in oncogenic signaling to promote tumor cell survival in epithelial tumors. Importantly, ROR1 is not expressed on vital organs, except adipose and pancreatic tissue, which reduces potential toxicities from killing of normal cells (Hudecek et al, 2013). ROR1 is expressed during embryogenesis but absent from normal adult tissues, apart from a subset of immature B-cell precursors, and low-level expression on adipocytes (Hudecek et al., 2010; Matsuda et al., 2001). ROR1 was first shown to be expressed in B-cell chronic lymphocytic leukemia (B-CLL) by transcriptional profiling (Klein et al., 2001; Rosenwald et al., 2001) and was subsequently identified on the surface of many cancers including mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL) with a t(1;19) chromosome translocation, and a subset of lung, breast, colon, pancreas, renal, and ovarian cancers (Baskar et al., 2008; Bicocca et al., 2012; Daneshmanesh et al., 2008; Dave et al., 2012; Fukuda et al., 2008; Yamaguchi et al., 2012; Zhang et al., 2012a, 2012b). In both lung adenocarcinoma and t(1;19) ALL, ROR1 cooperates in oncogenic signaling and knockdown of ROR1 with siRNA exposed a critical role for this molecule in maintaining tumor cell survival (Bicocca et al., 2012; Choudhury et al., 2010; Gentile et al., 2011; Yamaguchi et al., 2012). Thus, ROR1 loss may not be readily tolerated by tumors making it an attractive candidate for CAR directed T-cell therapy that could be broadly applied. The present inventors have thus considered that ROR1 could be a valuable target antigen for treating CLL as well as solid tumors such as breast, colon, lung, ovarian and kidney tumors, by using CAR-expressing T cells.

The laboratories of Dr. Stanley Riddell and Dr. Laurence Cooper have previously engineered and validated anti-ROR1 scCARs containing the 4A5 and the 2A2 scFvs, respectively (Cooper et al 2010; Hudecek et al., 2013). In particular, Hudecek et al discloses anti-ROR1 scCARs which contain an IgG4 hinge of diverse length and a CD28 transmembrane domain.

There is still the need for the improvement of CAR functionality by designing CAR architecture and using suitable components since these parameters play a role important and a fine tuning is necessary.

Therefore, as an alternative to the previous strategies, the present invention provides with ROR1 specific CARs, which can be expressed in immune cells to target ROR1 malignant cells with significant clinical advantage. The inventors have found that, by combining CAR architecture to the choice of suitable components, they could obtain specific ROR1 single chain CARs with high cytotoxicity towards cancerous target cells,

SUMMARY OF THE INVENTION

The inventors have generated ROR1 specific CAR having different structure and comprising different scFV derived from different ROR1 specific antibodies.

A ROR1 (NTRKR1) specific chimeric antigen receptor (CAR) according to the present invention may have one of the polypeptide structure selected from V1 to V6 as illustrated in FIG. 4, said structure comprising an extra cellular ligand binding-domain comprising VH and VL from a monoclonal anti-ROR1 antibody, a hinge, a transmembrane domain and a cytoplasmic domain including a CD3 zeta signaling domain and a co-stimulatory domain from 4-1BB.

More particularly, a ROR1 (NTRKR1) specific chimeric antigen receptor (CAR) according to the present invention may have one of the one of the polypeptide structure selected from V3, V5 and V1 as illustrated in FIG. 4, said structure comprising an extra cellular ligand binding-domain comprising VH and VL from a monoclonal anti-ROR1 antibody, a hinge, a CD8α transmembrane domain, a cytoplasmic domain including a CD3 zeta signaling domain and a co-stimulatory domain from 4-1BB. Such ROR1 (NTRKR1) specific antigen receptors have been found to have unexpected superior effects in term of cytotoxicity.

In an embodiment, a ROR1 specific CAR has a structure V1 comprises a FcγRIIIα hinge and CD8α transmembrane domain.

In a preferred embodiment, a ROR1 specific CAR has a structure V3 and comprises a CD8α hinge and a CD8α transmembrane domain.

In another preferred embodiment, a ROR1 specific CAR has a structure V5 comprises an IgG1 hinge and a CD8α transmembrane domain.

In particular, said antigen binding domains (scFvs) derived from H10 and D10 anti-ROR1 antibodies have shown remarkable anticancerous properties.

Moreover, humanized scFvs have been generated from the murine H10 and D10 anti-ROR1 antibodies.

Preferred CAR polypeptides of the invention comprise an amino acid sequence selected from SEQ ID NO. 79 to 138. Following non-specific activation in vitro (e.g. with anti CD3/CD28 coated beads and recombinant IL2), T-cells from donors have been transformed with polynucleotides expressing these CARs using viral transduction. In certain instances, the T-cells were further engineered to create non-alloreactive T-cells, more especially by disruption of a component of TCR (αβ—T-Cell receptors) to prevent Graft versus host reaction. The CAR of the present invention have been found particularly efficient in the context of allogeneic T-cells.

The resulting engineered T-cells displayed reactivity in-vitro against ROR1 positive cells to various extend, showing that the CARs of the present invention contribute to antigen dependent activation, and also proliferation, of the T-cells, making them useful for immunotherapy.

The polypeptides and polynucleotide sequences encoding the CARs of the present invention are detailed in the present specification.

The engineered immune cells of the present invention are particularly useful for therapeutic applications, such as for treating chronic lymphocytic leukemia (CLL), the Small Lymphocytic Lymphoma (SLL), the Mantle Cell Lymphoma (MCL), Acute Lymphoblastic Leukemia (ALL) with a t(1;19) chromosome translocation. They also can be used for treating solid tumors such as breast, colon, lung, and kidney tumors.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Single chain chimeric antigen receptors (scCARs) are most commonly created by joining the heavy- and light-chain variable regions of a monoclonal antibody (FIG. 1A) that binds to a specific antigen to the intracellular portion of a T cell signaling molecule, such as components of the TCR-associated CD3 complex (ζ

chain) (FIG. 1B). First generation of scars only contain a T cell signaling domain that transmits the activation signal (FIG. 1C left). Second generation scCARs incorporate in addition a single costimulatory molecule endodomain, such as the endodomain of CD28 or 41BB (FIG. 1C middle), that enhances T cell persistence and anti-tumor function in vivo. Third-generation scCARs incorporate at least two costimulatory molecule endodomains, such as the endodomain of CD28 and 41BB (FIG. 1C right).

FIG. 2: Structure of the ROR1 protein which is composed of extracellular part, transmembrane part, and intracellular domains; the extra and intracellular ones containing multiple parts as explained previously.

FIGS. 3A, 3B, 3C and 3D: (I) Alignment of sequences from D10 and H10 VH and VL murine sequences with germline human sequences encoded by V and J genes encoding the variable region of immunoglobulins, those sequence sharing high homology with murine sequences. For stability purpose (in particularly of CDRs), “the most critical” positions of AA are shown by arrows in the upper line correspond to AA which should be from murine origin, and the “less critical” ones in the lower line are those which can be either of human or murine origin. (II) MGT/DomainGapAlign (Lefranc et al. Dev. Comp. Immunol., 29, 185-203 (2005)) allows to align and “IMGT-gap” the domain amino acid sequence of D10 and H10 VH and VL murine sequences. The input sequence is displayed, aligned with the closest germline V-REGION or closest C-DOMAIN of the IMGT domain directory and with gaps according to the IMGT unique numbering for V-REGION, C-DOMAIN and C-LIKE-DOMAIN.

FIG. 4: schematic representation of the different CAR Architecture (V1 to V6).

FIG. 5: Number of ROR1 surface molecules on different human cell lines.

FIG. 6: scCARs screening procedure

FIG. 7: Total expression of scCARs in human T cells. (A) Experiment 1. (B) Experiment 2.

FIG. 8: Cell surface expression of scCARs on human T cells. (A) Experiment 1. (B) Experiment 2.

FIG. 9: Degranulation of scCAR modified T cells upon co-culture with target cells. (A) Experiment 1. (B) Experiment 2 (cell lines and/or treatment from the left to the right: MDA-MB-2301, PC-3, MCF-7, no activation, PMA+ ionomycin).

FIG. 10: Cell surface expression of scCARs on human T cells. Data are presented as mean+/−SD of four independent experiments.

FIG. 11: Degranulation of scCAR modified T cells upon co-culture with target cells. Data are presented as mean+/−SD of four independent experiments.

FIG. 12: IFNγ production by scCAR-modified T cells upon co-culture with target cells. Data are presented as mean+/−SD of four independent experiments (cell lines and/or treatment from the left to the right: Jeko-1, K562, MDA-MB-2301, PC-3, MCF-7, no activation).

FIG. 13: Cytotoxic activity of scCAR-modified T cells upon co-culture with adherent target cells. Data are presented as mean+/−SD of four independent experiments.

FIG. 14: Cytotoxic activity of scCAR-modified T cells upon co-culture with suspension target cells. Data are presented as mean+/−SD of four independent experiments.

FIG. 15: Schematic representation of an engineered immune cell according to the invention. The engineered immune cell presented in this figure is a T-cell transduced with a retroviral polypeptide encoding CAR. This T-cell is further engineered to allow a better and safer engraftment into the patient, which is optional within the frame of the present invention. X gene may be for instance a gene expressing a component of TCR (TCRalpha or TCRbeta), Y may be a gene involved into the sensitivity of T-cells to immune-suppressive drugs such as CD52 (with respect to alemtuzumab) or HPRT (with respect to 6-Thioguanine).

TABLE 1 Sequence of the different CAR components other than scFvs Functional domains SEQ ID # Raw amino acid sequence CD8α signal peptide SEQ ID NO. 1 MALPVTALLLPLALLLHAARP Alternative signal peptide SEQ ID NO. 2 METDTLLLWVLLLWVPGSTG Fcϵc SEQ ID NO. 3 GLAVSTISSFFPPGYQ CD8α SEQ ID NO. 4 TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGA VHTRGLDFACD IgG1 hinge SEQ ID NO. 5 EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDT LMIARTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK CD8α SEQ ID NO. 6 IYIWAPLAGTCGVLLLSLVITLYC 41BB transmembrane domain SEQ ID NO. 7 IISFFLALTSTALLFLLFFLTLRFSVV 41BB intracellular domain SEQ ID NO. 8 KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPE EEEGGCEL CD3ζintracellular domain SEQ ID NO. 9 RVKFSRSADAPAYQQGQNQLYNELNLGRREEY DVLDKRRGRDPEMGGKPRRKNPQEGLYNELQK DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA TKDTYDALHMQALPPR Linker SEQ ID NO. 10 GGGGSGGGGSGGGGS

TABLE 2 Sequence of the scFvs from murine origin, their CDRs of the scFv and humanized scFvs from D10 and H10 ScFv sequences SEQ ID # Raw amino acid sequence MURINE ORIGIN 2A2 heavy chain SEQ ID NO. 11 QVQLQQSGAELVRPGASVTLSCKASGYTFSDYEMHWVIQTPVHGLEWI variable region GAIDPETGGTAYNQKFKGKAILTADKSSSTAYMELRSLTSEDSAVYYCTGY YDYDSFTYWGQGTLVTVSA SEQ ID NO. 12 CDR1 GYTFSDYE SEQ ID NO. 13 CDR2 IDPETGGT SEQ ID NO. 14 CDR3 TGYYDYDSFTY 2A2 light chain SEQ ID NO. 15 DIVMTQSQKIMSTTVGDRVSITCKASQNVDAAVAWYQQKPGQSPKLLI variable region YSASNRYTGVPDRFTGSGSGTDFTLTISNMQSEDLADYFCQQYDIYPYTF GGGTKLEIK SEQ ID NO. 16 CDR1 QNVDAA SEQ ID NO. 17 CDR2 SAS SEQ ID NO. 18 CDR3 QQYDIYPYT 4A5 heavy chain SEQ ID NO. 19 EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQIPEKRLEWVA variable region SISRGGTTYYPDSVKGRFTISRDNVRNILYLQMSSLRSEDTAMYYCGRYD YDGYYAMDYWGQGTSVTVSS SEQ ID NO. 20 CDR1 GFTFSSYA SEQ ID NO. 21 CDR2 ISRGGTT SEQ ID NO. 22 CDR3 GRYDYDGYYAMDY 4A5 light chain SEQ ID NO. 23 DIKMTQSPSSMYASLGERVTITCKASPDINSYLSWFQQKPGKSPKTLIYRA variable region NRLVDGVPSRFSGGGSGQDYSLTINSLEYEDMGIYYCLQYDEFPYTFGGG TKLEMK SEQ ID NO. 24 CDR1 PDINSY SEQ ID NO. 25 CDR2 RAN SEQ ID NO. 26 CDR3 LQYDEFPYT D10 heavy chain SEQ ID NO. 27 QVQLKESGPGLVAPSQTLSITCTVSGFSLTSYGVHWVRQPPGKGLEWLG variable region VIWAGGFTNYNSALKSRLSISKDNSKSQVLLKMTSLQTDDTAMYYCARR GSSYSMDYWGQGTSVTVSS SEQ ID NO. 28 CDR-H1 GFSLTSYG SEQ ID NO. 29 CDR-H2 IWAGGFT SEQ ID NO. 30 CDR-H3 ARRGSSYSMDY D10 light chain SEQ ID NO. 32 EIVLSQSPAITAASLGQKVTITCSASSNVSYIHWYQQRSGTSPRPWIYEISK variable region LASGVPVRFSGSGSGTSYSLTISSMEAEDAAIYYCQQWNYPLITFGSGTKL EIQ SEQ ID NO. 33 CDR-L1 SNVSY SEQ ID NO. 34 CDR-L2 EIS SEQ ID NO. 35 CDR-L3 QQWNYPLIT G6 heavy chain SEQ ID NO. 37 EVQLQQSGPELEKPGASVKISCKASGFAFTGYNMNWVKQTNGKSLEWI variable region GSIDPYYGGSTYNQKFKDKATLTVDKSSSTAYMQLKSLTSDDSAVYYCAR SPGGDYAMDYWGQGTSVTVSS SEQ ID NO. 38 CDR1 GFAFTGYN SEQ ID NO. 39 CDR2 IDPYYGGS SEQ ID NO. 40 CDR3 ARSPGGDYAMDY G6 light chain SEQ ID NO. 41 DIKMTQSPSSMYASVGERVTITCKASQGINSYSGWFQQKPGKSPKTLIYR variable region GNRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPYTFGG GTKLEIK SEQ ID NO. 42 CDR1 QGINSY SEQ ID NO. 43 CDR2 RGN SEQ ID NO. 44 CDR3 LQYDEFPYT G3 heavy chain SEQ ID NO. 45 QVQLQQPGAELVKPGTSVKLSCKASGYNFTNYWINWVKLRPGQGLEWI variable region GEIYPGSGSTNYNEKFKSKATLTADTSSSTAYMQLSSLASEDSALYYCARD GNYYAMDYWGQGTSVTVSS SEQ ID NO. 46 CDR1 GYNFTNYW SEQ ID NO. 47 CDR2 IYPGSGST SEQ ID NO. 48 CDR3 ARDGNYYAMDY G3 light chain SEQ ID NO. 49 DIQMTQTTSSLSASLGDRVTITCRASQDINNYLNWYQQKPDGTVKLLIYY variable region TSALHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPPYTFGG GTKLEIK SEQ ID NO. 50 CDR1 QDINNY SEQ ID NO. 51 CDR2 YTS SEQ ID NO. 52 CDR3 QQGNTLPPYT H10 heavy chain SEQ ID NO. 53 EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPEKRLEWVA variable region SISTGASAYFPDSVKGRFTISRDNARNILYLQMSSLRSEDTAMYYCARITT STWYFDVWGAGTTVTVSS SEQ ID NO. 54 CDR1-H1 GFTFSSYA SEQ ID NO. 55 CDR-H2 ISTGASA SEQ ID NO. 56 CDR-H3 ARITTSTWYFDV H10 light chain SEQ ID NO. 58 DIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYR variable region ANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPYTFGG GTKLEIK SEQ ID NO. 59 CDR-L1 QDINSY SEQ ID NO. 60 CDR-L2 RAN SEQ ID NO. 61 CDR-L3 LQYDEFPYT 2A4 heavy chain SEQ ID NO. 63 EVKLQQSGPELVKPGASVKISCKTSGYTFTEYTMHWVKQSHGKSLEWIG variable region GINPNNGGTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCALQ GFAYWGQGTPLTVSS SEQ ID NO. 64 CDR1 GYTFTEYT SEQ ID NO. 65 CDR2 INPNNGGT SEQ ID NO. 66 CDR3 ALQGFAY 2A4 light chain SEQ ID NO. 67 MEIEITQTPALMSASPGEKVTMTCSASSSVSYMYWYQQKPRSSPKPWIY variable region LTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPYTFG GGTRLELK SEQ ID NO. 68 CDR1 SSVSY SEQ ID NO. 69 CDR2 LTS SEQ ID NO. 70 CDR3 QQWSSNPYT 1C11 heavy chain SEQ ID NO. 71 EVKLQESGAELARPGASVKMSCKASGYTFTSYTMHWVKQRPGQGLEWI variable region GYINPSSGYTEYNQKFKDKTTLTADKSSSTAYMQLSSLTSGDSAVYYCAR RVLWLRRGDYWGQGTILTVSA SEQ ID NO. 72 CDR1 GYTFTSYT SEQ ID NO. 73 CDR2 INPSSGYT SEQ ID NO. 74 CDR3 ARRVLWLRRGDY 1C11 light chain SEQ ID NO. 75 MEVLITQTPSSLSASLGERVSLTCRASQDIGSSLNWLQQEPDGTIKRLIYA variable region TSSLDSGVPKRFSGSRSGSDYSLTISSLESEDFVDYYCLQYASSPYTFGGGT KLELK SEQ ID NO. 76 CDR1 QDIGSS SEQ ID NO. 77 CDR2 ATS SEQ ID NO. 78 CDR3 LQYASSP HUMANIZED For the VH chain SEQ ID NO. 31 QVQL(Q/K)ESGPGLV(K/A)PSETLSLICTVSGFSLTSYGVHWVRCIPPGK of D10 antibody GLEWLGVIWAGGFTNYN(P/S)SLKSRLTISKDNSKNQVSLKLSSVTA(A/D) Hu D10-VH DTA(V/M)YYCARRGSSYSMDYWGQGTLVTVSS For the VL chain SEQ ID NO. 36 EIVL(T/S)QSPATLSLSPGERATLSC(R/S)ASSNVSYIHWYQQK(P/S)GQA of D10 antibody PRPWIYEISKLA(T/S)GIPARFSGSGSGTDYTLTISSLE(P/A)EDFA(V/I)YY Hu D10-VL CQQWNYPLITFGQGTKLEIK For the VH chain SEQ ID NO. 57 EV(Q/K)LVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLE of H10 antibody WVASISTGASAYF(A/P)DSVKGRFTISRDNAKNSLYLQMNSLRAEDTA(V/M) Hu H10-VH YYCARITTSTWYFDVWGQGTTVTVSS For the VL chain SEQ ID NO. 62 DI(Q/K)MTQSPSSLSASVGDRVTITCRASQDINSYLSWFQQKPGKAPKTL of H10 antibody IYRANRL(Q/V)SGVPSRFSGSGSGQDYTLTISSLQ(P/Y)EDFATYYCLQYD Hu H10-VL EFPYTFGQGTKLEIK

TABLE 3 CAR of structure V-1 CAR Structure CAR signal Designation peptide FcγRIIIα V-1 (optional) VH VL hinge CD8α-TM 41BB-IC CD3ζ CD 2A2-v1 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 79) NO. 1 NO. 11 NO. 15 NO. 3 NO. 6 NO. 8 NO. 9 4A5-v1 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 85) NO. 1 NO. 19 NO. 23 NO. 3 NO. 6 NO. 8 NO. 9 D10-v1-murine SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID & humanized NO. 1 NO. 27 or 31 NO. 32 or NO. 3 NO. 6 NO. 8 NO. 9 (SEQ ID NO. 91 36 and 97) G6-v1 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 1 NO. 37 NO. 41 NO. 3 NO. 6 NO. 8 NO. 9 NO. 103) G3-v1 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 1 NO. 45 NO. 49 NO. 3 NO. 6 NO. 8 NO. 9 NO. 109) H10-v1 murine SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID & humanized NO. 1 NO. 53 or 57 NO. 58 or NO. 3 NO. 6 NO. 8 NO. 9 (SEQ ID 62 NO. 115 and 121) 2A4-v1 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 1 NO. 63 NO. 67 NO. 3 NO. 6 NO. 8 NO. 9 NO. 127) 1C11-v1 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 1 NO. 71 NO. 75 NO. 3 NO. 6 NO. 8 NO. 9 NO. 133)

TABLE 4 CAR of structure V-2 CAR Structure CAR signal Designation peptide FcγRIIIα V-2 (optional) VH VL hinge 41BB-TM 41BB-IC CD3ζCD 2A2-v2 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 80) NO. 1 NO. 11 NO. 15 NO. 3 NO. 7 NO. 8 NO. 9 4A5-v2 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 86) NO. 1 NO. 19 NO. 23 NO. 3 NO. 7 NO. 8 NO. 9 D10-v2 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID murine & NO. 1 NO. 27 or 31 NO. 32 or NO. 3 NO. 7 NO. 8 NO. 9 humanized 36 (SEQ ID NO. 92 and 98) G6-v2 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 1 NO. 37 NO. 41 NO. 3 NO. 7 NO. 8 NO. 9 NO. 104) G3-v2 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 1 NO. 45 NO. 49 NO. 3 NO. 7 NO. 8 NO. 9 NO. 110) H10-v2 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID murine & NO. 1 NO. 53 or 57 NO. 58 or NO. 3 NO. 7 NO. 8 NO. 9 humanized 62 (SEQ ID NO. 116 and 122) 2A4-v2 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 1 NO. 63 NO. 67 NO. 3 NO. 7 NO. 8 NO. 9 NO. 128) 1C11-v2 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 1 NO. 71 NO. 75 NO. 3 NO. 7 NO. 8 NO. 9 NO. 134)

TABLE 5 CAR of structure V-3 CAR Structure CAR signal Designation peptide V-3 (optional) VH VL CD8α hinge CD8α-TM 41BB-IC CD3ζCD 2A2-v3 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 81) NO. 1 NO. 11 NO. 15 NO. 4 NO. 6 NO. 8 NO. 9 4A5-v3 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 87) NO. 1 NO. 19 NO. 23 NO. 4 NO. 6 NO. 8 NO. 9 D10-v3 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID murine & NO. 1 NO. 27 or 31 NO. 32 or NO. 4 NO. 6 NO. 8 NO. 9 humanized 36 (SEQ ID NO. 93 and 99)) G6-v3 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 1 NO. 37 NO. 41 NO. 4 NO. 6 NO. 8 NO. 9 NO. 105) G3-v3 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 1 NO. 45 NO. 49 NO. 4 NO. 6 NO. 8 NO. 9 NO. 111) H10-v3 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID murine & NO. 1 NO. 53 or 57 NO. 58 or NO. 4 NO. 6 NO. 8 NO. 9 humanized 62 (SEQ ID NO. 117 and 123) 2A4-v3 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 1 NO. 63 NO. 67 NO. 4 NO. 6 NO. 8 NO. 9 NO. 129) 1C11-v3 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 1 NO. 71 NO. 75 NO. 4 NO. 6 NO. 8 NO. 9 NO. 135)

TABLE 6 CAR of structure V-4 CAR Structure CAR signal Designation peptide V-4 (optional) VH VL CD8α hinge 41BB-TM 41BB-IC CD3ζCD 2A2-v4 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 82) NO. 1 NO. 11 NO. 15 NO. 4 NO. 7 NO. 8 NO. 9 4A5-v4 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 88) NO. 1 NO. 19 NO. 23 NO. 4 NO. 7 NO. 8 NO. 9 D10-v4 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID murine & NO. 1 NO. 27 or 31 NO. 32 or NO. 4 NO. 7 NO. 8 NO. 9 humanized 36 (SEQ ID NO. 94 and 100) G6-v4 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 1 NO. 37 NO. 41 NO. 4 NO. 7 NO. 8 NO. 9 NO. 106) G3-v4 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 1 NO. 45 NO. 49 NO. 4 NO. 7 NO. 8 NO. 9 NO. 112) H10-v4 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID murine & NO. 1 NO. 53 or 57 NO. 58 or NO. 4 NO. 7 NO. 8 NO. 9 humanized 62 (SEQ ID NO. 118 and 124) 2A4-v4 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 1 NO. 63 NO. 67 NO. 4 NO. 7 NO. 8 NO. 9 NO. 130) 1C11-v4 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 1 NO. 71 NO. 75 NO. 4 NO. 7 NO. 8 NO. 9 NO. 136)

TABLE 7 CAR of structure V-5 CAR Structure CAR signal Designation peptide V-5 (optional) VH VL IgG1 hinge CD8α-TM 41BB-IC CD3ζCD 2A2-v5 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 83) NO. 1 NO. 11 NO. 15 NO. 5 NO. 6 NO. 8 NO. 9 4A5-v5 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 89) NO. 1 NO. 19 NO. 23 NO. 5 NO. 6 NO. 8 NO. 9 D10-v5 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID murine & NO. 1 NO. 27 or 31 NO. 32 or NO. 5 NO. 6 NO. 8 NO. 9 humanized 36 (SEQ ID NO. 95 and 101) G6-v5 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 1 NO. 37 NO. 41 NO. 5 NO. 6 NO. 8 NO. 9 NO. 107) G3-v5 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 1 NO. 45 NO. 49 NO. 5 NO. 6 NO. 8 NO. 9 NO. 113) H10-v5 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID murine & NO. 1 NO. 53 or 57 NO. 58 or NO. 5 NO. 6 NO. 8 NO. 9 humanized 62 (SEQ ID NO. 119 and 125) 2A4-v5 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 1 NO. 63 NO. 67 NO. 5 NO. 6 NO. 8 NO. 9 NO. 131) 1C11-v5 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 1 NO. 71 NO. 75 NO. 5 NO. 6 NO. 8 NO. 9 NO. 137)

TABLE 8 CAR of structure V-6 CAR Structure CAR signal Designation peptide V-6 (optional) VH VL IgG1 hinge 41BB-TM 41BB-IC CD3ζCD 2A2-v6 SEQ ID VH VL SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 84) NO. 1 NO. 5 NO. 7 NO. 8 NO. 9 4A5-v6 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 90) NO. 1 NO. 11 NO. 15 NO. 5 NO. 7 NO. 8 NO. 9 D10-v6 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID murine & NO. 1 NO. 19 NO. 23 NO. 5 NO. 7 NO. 8 NO. 9 humanized (SEQ ID NO. 96 and 102) G6-v6 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 1 NO. 27 or 31 NO. 32 or NO. 5 NO. 7 NO. 8 NO. 9 NO. 108) 36 G3-v6 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 1 NO. 37 NO. 41 NO. 5 NO. 7 NO. 8 NO. 9 NO. 114) H10-v6 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID murine & NO. 1 NO. 45 NO. 49 NO. 5 NO. 7 NO. 8 NO. 9 humanized (SEQ ID NO. 120 and 126) 2A4-v6 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 1 NO. 53 or 57 NO. 58 or NO. 5 NO. 7 NO. 8 NO. 9 NO. 132) 62 1C11-v6 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 1 NO. 63 NO. 67 NO. 5 NO. 7 NO. 8 NO. 9 NO. 138)

DETAILED DESCRIPTION OF THE INVENTION

Unless specifically defined herein, all technical and scientific terms used have the same meaning as commonly understood by a skilled artisan in the fields of gene therapy, biochemistry, genetics, and molecular biology.

All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, with suitable methods and materials being described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will prevail. Further, the materials, methods, and examples are illustrative only and are not intended to be limiting, unless otherwise specified.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Harries & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J. Abelson and M. Simon, eds.-in-chief, Academic Press, Inc., New York), specifically, Vols. 154 and 155 (Wu et al. eds.) and Vol. 185, “Gene Expression Technology” (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

ROR1 Specific Chimeric Antigen Receptors

The present invention relates to new designs of anti-ROR1 chimeric antigen receptor (CAR) comprising an extracellular ligand-binding domain, a transmembrane domain and a signaling transducing domain.

The term “extracellular ligand-binding domain” as used herein is defined as an oligo- or polypeptide that is capable of binding a ligand. Preferably, the domain will be capable of interacting with a cell surface molecule. For example, the extracellular ligand-binding domain may be chosen to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state. In a preferred embodiment, said extracellular ligand-binding domain comprises a single chain antibody fragment (scFv) comprising the light (V_(L)) and the heavy (V_(H)) variable fragment of a target antigen specific monoclonal anti CD-123 antibody joined by a flexible linker. Said V_(L) and V_(H) are preferably selected from the antibodies referred to as 2A2, 4A5 and D10 as indicated in Table 2.

Moreover, Table 2 referring also to V_(L) and V_(H) of G6, G3, H10, 2A4 and 1C11, discloses their respective sequences SEQ ID NO. 37, 41, 45, 49, 53, 58, 63, 67, 71 and 75, as well as humanized V_(L) and V_(H) of D10 and H10 of respective sequence SEQ ID NO. 31, 36, 57 and 62.

FIG. 4 shows the architecture of the 6 versions of CARs according to the present invention. Table 1 shows, as examples, the components used in the further experiments.

A ROR1 (NTRKR1) specific chimeric antigen receptor (CAR) according to the present invention may have one of the polypeptide structure selected from V1 to V6 as illustrated in FIG. 4, said structure comprising an extra cellular ligand binding-domain comprising VH and VL from a monoclonal anti-ROR1 antibody, a hinge, a transmembrane domain and a cytoplasmic domain including a CD3 zeta signaling domain and a co-stimulatory domain from 4-1BB.

More specifically, the present invention relates to ROR1 (NTRKR1) specific chimeric antigen receptors (CARs) having one of the polypeptide structure selected from V3, V5 and V1 as illustrated in FIG. 4, said structure comprising an extra cellular ligand binding-domain comprising VH and VL from a monoclonal anti-ROR1 antibody, a hinge, a CD8α transmembrane domain and a cytoplasmic domain including a CD3 zeta signaling domain and a co-stimulatory domain from 4-1BB.

Antigen-Binding Domain

The antigen binding domain of the ROR1 CARs of the invention can be any domain that binds to the off-tissue antigen including but not limited to a monoclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, and a functional fragment thereof.

In a preferred embodiment, said extracellular ligand-binding domain comprises a single chain antibody fragment (scFv) comprising the light (V_(L)) and the heavy (V_(H)) variable fragment of a target antigen specific monoclonal ROR1 antibody joined by a flexible linker. Said V_(L) and V_(H) are preferably selected from the antibodies referred to as H10, D10, 4A5, G6, G3, 2A2, 2A4, and 1C11 as indicated in Table 2. They are preferably linked together by a flexible linker comprising for instance the sequence SEQ ID NO:10. In other words, said CARs preferentially comprise an extracellular ligand-binding domain comprising a polypeptide sequence displaying at least 90%, 95% 97% or 99% identity with sequences issued from combinations of VH and VL chains—with a linker between them—as depicted in Table 3 to Table 8 for versions V1 to V6.

In particular preferred embodiments, said V_(L) and V_(H) are derived from the antibody H10.

In other particular embodiments, said V_(L) and V_(H) are derived from the antibody D10.

By the term “recombinant antibody” as used herein, is meant an antibody or antibody fragment which is generated using recombinant DNA technology, such as, for example, an antibody or antibody fragment expressed by a bacteriophage, a yeast expression system or a mammalian cell expression system, and more especially by a T cell transduced with a viral vector comprising a nucleic acid sequence encoding CDR regions of an antibody. The term should also be construed to mean an antibody or antibody fragment which has been generated by the synthesis of a DNA molecule encoding the antibody or antibody fragment and which DNA molecule expresses an antibody or antibody fragment protein, or an amino acid sequence specifying the antibody or antibody fragment, wherein the DNA or amino acid sequence has been obtained using recombinant or synthetic DNA or amino acid sequence technology which is available and well known in the art.

By the term “humanized antibody” as used herein, is meant the polypeptides include a humanized heavy chain variable region and a humanized light chain variable region. For example, the polypeptides may include the framework (FR) regions of the light and heavy chain variable regions of a human antibody, while retaining substantially the antigen-binding specificity of a parental monoclonal antibody. The humanized heavy chain variable region and/or the humanized light chain variable region are at least about 87% humanized, at least about 90% humanized, at least about 95% humanized, at least about 98% humanized, or at least about 100% humanized, excluding the complementary-determining regions (CDRs). The antigen-binding polypeptides molecules may be derived from monoclonal antibody donors (e.g., mouse monoclonal antibody donors) and may include CDRs from the monoclonal antibodies (e.g., mouse monoclonal CDRs). When

By the term “monoclonal antibody” as used herein, is meant antibody produced by a laboratory-grown cell clone, either of a hybridoma or a virus-transformed lymphocyte, that is more abundant and uniform than natural antibody and is able to bind specifically to a single site on ROR1 antigen. They are monospecific antibodies that are made by identical immune cells that are all clones of a unique parent cell, in contrast to polyclonal antibodies which are made from several different immune cells. Monoclonal antibodies have monovalent affinity, in that they bind to the same epitope.

The present invention discloses a ROR1 specific chimeric antigen receptor (ROR1 CAR) as above, wherein said extra cellular ligand binding-domain comprises VH and VL chains which are humanized.

Table 2 shows the sequences of humanized scFv (VH and VL chains) corresponding to the D10 and H10 anti-ROR1 antibodies.

FIGS. 3A to 3D present the alignments of D10 and H10 scFvs of murine versus their humanized form after having applied the methodology according to Lefranc M P et al (Lefranc, M P, Ehrenmann F, Ginestoux C, Giudicelli V, Duroux P “Use of IMGT (®) databases and tools for antibody engineering and humanization”, Methods Mol Biol. 2012; 907: 3-37). In these four alignments are indicated:

-   -   the CDRs,     -   the “most critical” amino-acids (AAs) with a upper arrow which         mean that those AAs are maintained as murine origin;     -   the “less critical” amino-acids with a upper arrow which mean         that those AAs can be of murine origin or human origin; it is         understood that the humanized sequences of the invention are         actually a set of all the different sequences possible made by         all the combinations of such “less critical” AAs from human or         murine origin; those sequences containing the CDRs and “most         critical” AAs as presented just previously.

A humanized antibody can be produced using a variety of techniques known in the art, including but not limited to, CDR-grafting (see, e.g., European Patent No. EP 239,400; International Publication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089, each of which is incorporated herein in its entirety by reference), veneering or resurfacing (see, e.g., European Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnicka et al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al., 1994, PNAS, 91:969-973, each of which is incorporated herein by its entirety by reference), chain shuffling (see, e.g., U.S. Pat. No. 5,565,332, which is incorporated herein in its entirety by reference), and techniques disclosed in, e.g., U.S. Patent Application Publication No. US2005/0042664, U.S. Patent Application Publication No. US2005/0048617, U.S. Pat. Nos. 6,407,213, 5,766,886, International Publication No. WO 9317105, Tan et al., J. Immunol., 169: 1119-25 (2002), Caldas et al., Protein Eng., 13(5):353-60 (2000), Morea et al., Methods, 20(3):267-79 (2000), Baca et al., J. Biol. Chem., 272(16): 10678-84 (1997), Roguska et al., Protein Eng., 9(10):895-904 (1996), Couto et al., Cancer Res., 55 (23 Supp):5973s-5977s (1995), Couto et al., Cancer Res., 55(8): 1717-22 (1995), Sandhu J S, Gene, 150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol., 235(3):959-73 (1994), each of which is incorporated herein in its entirety by reference. Often, framework residues in the framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, for example improve, antigen binding. These framework substitutions are identified by methods well-known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature, 332:323, which are incorporated herein by reference in their entireties.).

One aspect of the present invention relates particularly to a ROR1 (NTRKR1) specific chimeric antigen receptor (CAR) having one of the polypeptide structure selected from V3, V5 and V1 as illustrated in FIG. 4, said structure comprising an extra cellular ligand binding-domain comprising VH and VL from a monoclonal anti-ROR1 antibody, a hinge, a CD8α transmembrane domain (CD8α TM) and a cytoplasmic domain including a CD3 zeta signaling domain and a co-stimulatory domain from 4-1BB.

According to a preferred embodiment, said transmembrane domain is encoded by a polypeptide which shares at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity with SEQ ID NO. 6 (CD8α TM).

According to another preferred embodiment, said hinge is encoded by a polypeptide which shares at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity, respectively for the structures V3, V5 and V1, with SEQ ID NO. 4 (CD8α), SEQ ID NO. 5 (IgG1) and SEQ ID NO. 3 (FcγRIIIα).

According to a more preferred embodiment, the ROR1-specific CAR of the invention has the polypeptide structure V3 comprising a CD8α hinge having the amino acid sequence set forth in SEQ ID NO. 4 and a CD8α transmembrane domain having the amino acid sequence set forth in SEQ ID NO:6.

According to another more preferred embodiment, the ROR1 specific CAR of the invention has the polypeptide structure V5 comprises IgG1 hinge having the amino acid sequence set forth in SEQ ID NO. 5 and a CD8α transmembrane domain having the amino acid sequence set forth in SEQ ID NO. 6.

According to an embodiment, said ROR1 (NTRKR1) specific chimeric antigen receptor (CAR) having one of the polypeptide structure selected from V3, V5 and V1 as illustrated in FIG. 4, wherein the extracellular ligand binding domain comprises:

-   -   a variable heavy chain VH comprising the CDRs from the mouse         monoclonal antibody H10 of SEQ ID NO. 54 (CDR-H1), SEQ ID NO:55         (CDR-H2) and SEQ ID NO:56 (CDR-H3), and;     -   a variable light VL comprising the CDRs from the mouse         monoclonal antibody H10 of SEQ ID NO. 59 (CDR-L1), SEQ ID NO:60         (CDR-L2) and SEQ ID NO:61 (CDR-L3);

or:

-   -   a variable heavy VH comprising the CDRs from the mouse         monoclonal antibody D10 of SEQ ID NO. 28 (CDR-H1), SEQ ID NO. 29         (CDR-H2) and SEQ ID NO. 30 (CDR-H3), and;     -   a variable light VL comprising the CDRs from the mouse         monoclonal antibody D10 of SEQ ID NO. 33 (CDR-L1), SEQ ID NO:34         (CDR-L2) and SEQ ID NO:35 (CDR-L3).

In a preferred embodiment, said ROR1 (NTRKR1) specific chimeric antigen receptor (CAR) having one of the polypeptide structure selected from V3, V5 and V1 as illustrated in FIG. 4, wherein said extra cellular ligand binding-domain comprising VH and VL chains which have at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity respectively with

-   -   SEQ ID NO:53 (H10-VH) and SEQ ID NO:58 (H10-VL), or     -   SEQ ID NO:27 (D10-VH) and SEQ ID NO:32 (D10-VL), or;

In another embodiment, said ROR1 (NTRKR1) specific chimeric antigen receptor (CAR) having one of the polypeptide structure selected from V3, V5 and V1 as illustrated in FIG. 4, wherein said extra cellular ligand binding-domain comprises VH and VL chains from H10 or D10 antibodies which have been humanized.

In particular, said cellular ligand binding-domain comprises humanized VH and VL chains, wherein,

-   -   a variable heavy Hu H10 VH has a polypeptide encoded by SEQ ID         NO: 57, and     -   a variable light Hu H10 VH has a polypeptide encoded by SEQ ID         NO: 62;

or:

-   -   a variable heavy Hu D10 VH has a polypeptide encoded by SEQ ID         NO: 31, and     -   a variable light Hu D10 has a polypeptide encoded by SEQ ID NO:         36.

According to another embodiment, said ROR1 (NTRKR1) specific chimeric antigen receptor (CAR) having one of the polypeptide structure selected from V3, V5 and V1 as illustrated in FIG. 4, wherein said CAR polypeptide shares at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity with SEQ ID NO:117 (H10v3-CAR sequence), or with SEQ ID NO:93 (D10v3-CAR sequence), or with SEQ ID NO:95 (D10v5-CAR sequence).

CAR Architectures

The signal transducing domain or intracellular signaling domain of a CAR according to the present invention is responsible for intracellular signaling following the binding of extracellular ligand binding domain to the target resulting in the activation of the immune cell and immune response. In other words, the signal transducing domain is responsible for the activation of at least one of the normal effector functions of the immune cell in which the CAR is expressed. For example, the effector function of a T cell can be a cytolytic activity or helper activity including the secretion of cytokines. Thus, the term “signal transducing domain” refers to the portion of a protein which transduces the effector signal and directs the cell to perform a specialized function.

Preferred examples of signal transducing domain for use in a CAR can be the cytoplasmic sequences of the T cell receptor and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivate or variant of these sequences and any synthetic sequence that has the same functional capability. Signal transducing domain comprises two distinct classes of cytoplasmic signaling sequence, those that initiate antigen-dependent primary activation, and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal. Primary cytoplasmic signaling sequence can comprise signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs. ITAMs are well defined signaling motifs found in the intracytoplasmic tail of a variety of receptors that serve as binding sites for syk/zap70 class tyrosine kinases. Examples of ITAM used in the invention can include as non-limiting examples those derived from TCRzeta, FcRgamma, FcRbeta, FcRepsilon, CD3gamma, CD3delta, CD3epsilon, CD5, CD22, CD79a, CD79b and CD66d. In a preferred embodiment, the signaling transducing domain of the CAR can comprise the CD3zeta signaling domain which has amino acid sequence with at least 70%, preferably at least 80%, more preferably at least 90%, 95% 97% or 99% sequence identity with amino acid sequence (SEQ ID NO: 9).

In particular embodiment the signal transducing domain of the CAR of the present invention comprises a co-stimulatory signal molecule or a part of it. A co-stimulatory molecule is a cell surface molecule other than an antigen receptor or their ligands that is required for an efficient immune response. “Co-stimulatory ligand” refers to a molecule on an antigen presenting cell that specifically binds a cognate co-stimulatory molecule on a T-cell, thereby providing a signal which, in addition to the primary signal provided by, for instance, binding of a TCR/CD3 complex with an MHC molecule loaded with peptide, mediates a T cell response, including, but not limited to, proliferation activation, differentiation and the like. A co-stimulatory ligand can include but is not limited to CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory igand (ICOS-L), intercellular adhesion molecule (ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, an agonist or antibody that binds Toll ligand receptor and a ligand that specifically binds with B7-H3. A co-stimulatory ligand also encompasses, inter alia, an antibody that specifically binds with a co-stimulatory molecule present on a T cell, such as but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LTGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83.

A “co-stimulatory molecule” refers to the cognate binding partner on a T-cell that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the cell, such as, but not limited to proliferation. Co-stimulatory molecules include, but are not limited to, an MHC class I molecule, BTLA and Toll ligand receptor. Examples of costimulatory molecules include CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 and a ligand that specifically binds with CD83 and the like.

In a preferred embodiment, the signal transducing domain of the CAR of the present invention comprises a co-stimulatory signal molecule or a part of it selected from the group consisting of fragment of 4-1BB (GenBank: AAA53133.) and CD28 (NP_006130.1). In particular the signal transducing domain of the CAR of the present invention comprises amino acid sequence which comprises at least 70%, preferably at least 80%, more preferably at least 90%, 95% 97% or 99% sequence identity with amino acid sequence selected from the group consisting of SEQ ID NO: 8.

A CAR according to the present invention is expressed on the surface membrane of the cell. Thus, such CAR further comprises a transmembrane domain. The distinguishing features of appropriate transmembrane domains comprise the ability to be expressed at the surface of a cell, preferably in the present invention an immune cell, in particular lymphocyte cells or Natural killer (NK) cells, and to interact together for directing cellular response of immune cell against a predefined target cell. The transmembrane domain can be derived either from a natural or from a synthetic source. The transmembrane domain can be derived from any membrane-bound or transmembrane protein. As non-limiting examples, the transmembrane polypeptide can be a subunit of the T-cell receptor such as α, β, γ or

, polypeptide constituting CD3 complex, IL2 receptor p55 (α chain), p75 (β chain) or γ chain, subunit chain of Fc receptors, in particular Fcγ receptor III or CD proteins. Alternatively the transmembrane domain can be synthetic and can comprise predominantly hydrophobic residues such as leucine and valine. In a preferred embodiment said transmembrane domain is derived from the human CD8 alpha chain (e.g. NP_001139345.1) The transmembrane domain can further comprise a hinge region between said extracellular ligand-binding domain and said transmembrane domain. The term “hinge region” used herein generally means any oligo- or polypeptide that functions to link the transmembrane domain to the extracellular ligand-binding domain. In particular, hinge region are used to provide more flexibility and accessibility for the extracellular ligand-binding domain. A hinge region may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. Hinge region may be derived from all or part of naturally occurring molecules, such as from all or part of the extracellular region of CD8, CD4 or CD28, or from all or part of an antibody constant region. Alternatively the hinge region may be a synthetic sequence that corresponds to a naturally occurring hinge sequence, or may be an entirely synthetic hinge sequence. In a preferred embodiment said hinge domain comprises a part of human CD8 alpha chain, FcγRIIIα receptor or IgG1 respectively referred to in this specification as SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5, or hinge polypeptides which display preferably at least 80%, more preferably at least 90%, 95% 97% or 99% sequence identity with these polypeptides.

A CAR according to the invention generally further comprises a transmembrane domain (TM) more particularly selected from CD8α and 4-1BB, showing identity with the polypeptides of SEQ ID NO. 6 or 7. The CD8α TM of SEQ ID NO. 6 is preferred.

According to preferred embodiments, the ROR1 specific CAR has a polypeptide structure selected from V3, V5 and V1 as illustrated in FIG. 4, said structure comprising an extra cellular ligand binding-domain comprising VH and VL from a monoclonal anti-ROR1 antibody, a CD8α hinge, a CD8α transmembrane domain, and a cytoplasmic domain including a CD3 zeta signaling domain and a co-stimulatory domain from 4-1BB.

According another preferred embodiment, the ROR1 specific CAR has a polypeptide structure selected from V3, V5 and V1 as illustrated in FIG. 4, said structure comprising an extra cellular ligand binding-domain comprising VH and VL from a monoclonal anti-ROR1 antibody, an IgG1 hinge, a CD8α transmembrane domain and a cytoplasmic domain including a CD3 zeta signaling domain and a co-stimulatory domain from 4-1BB.

According a preferred embodiment, the ROR1 specific CAR has a polypeptide structure selected from V3, V5 and V1 as illustrated in FIG. 4, said structure comprising an extra cellular ligand binding-domain comprising VH and VL from a monoclonal anti-ROR1 antibody, a FcγRIIIα hinge, a CD8α transmembrane domain, and a cytoplasmic domain including a CD3 zeta signaling domain and a co-stimulatory domain from 4-1BB.

Tables 3-8 show the CAR structures for all the 6 versions V1 to V6.

Downregulation or mutation of target antigens is commonly observed in cancer cells, creating antigen-loss escape variants. Thus, to offset tumor escape and render immune cell more specific to target, the ROR1 specific CAR according to the invention can comprise another extracellular ligand-binding domains, to simultaneously bind different elements in target thereby augmenting immune cell activation and function. In one embodiment, the extracellular ligand-binding domains can be placed in tandem on the same transmembrane polypeptide, and optionally can be separated by a linker. In another embodiment, said different extracellular ligand-binding domains can be placed on different transmembrane polypeptides composing the CAR. In another embodiment, the present invention relates to a population of CARs comprising each one different extracellular ligand binding domains. In a particular, the present invention relates to a method of engineering immune cells comprising providing an immune cell and expressing at the surface of said cell a population of CAR each one comprising different extracellular ligand binding domains. In another particular embodiment, the present invention relates to a method of engineering an immune cell comprising providing an immune cell and introducing into said cell polynucleotides encoding polypeptides composing a population of CAR each one comprising different extracellular ligand binding domains. By population of CARs, it is meant at least two, three, four, five, six or more CARs each one comprising different extracellular ligand binding domains. The different extracellular ligand binding domains according to the present invention can preferably simultaneously bind different elements in target thereby augmenting immune cell activation and function. The present invention also relates to an isolated immune cell which comprises a population of CARs each one comprising different extracellular ligand binding domains.

Polynucleotides, Vectors:

The present invention also relates to polynucleotides, vectors encoding the above described CAR according to the invention.

The polynucleotide may consist in an expression cassette or expression vector (e.g. a plasmid for introduction into a bacterial host cell, or a viral vector such as a baculovirus vector for transfection of an insect host cell, or a plasmid or viral vector such as a lentivirus for transfection of a mammalian host cell).

In a particular embodiment, the different nucleic acid sequences can be included in one polynucleotide or vector which comprises a nucleic acid sequence encoding ribosomal skip sequence such as a sequence encoding a 2A peptide. 2A peptides, which were identified in the Aphthovirus subgroup of picornaviruses, causes a ribosomal “skip” from one codon to the next without the formation of a peptide bond between the two amino acids encoded by the codons (see (Donnelly and Elliott 2001; Atkins, Wills et al. 2007; Doronina, Wu et al. 2008)). By “codon” is meant three nucleotides on an mRNA (or on the sense strand of a DNA molecule) that are translated by a ribosome into one amino acid residue. Thus, two polypeptides can be synthesized from a single, contiguous open reading frame within an mRNA when the polypeptides are separated by a 2A oligopeptide sequence that is in frame. Such ribosomal skip mechanisms are well known in the art and are known to be used by several vectors for the expression of several proteins encoded by a single messenger RNA.

To direct transmembrane polypeptide into the secretory pathway of a host cell, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in polynucleotide sequence or vector sequence. The secretory signal sequence is operably linked to the transmembrane nucleic acid sequence, i.e., the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell. Secretory signal sequences are commonly positioned 5′ to the nucleic acid sequence encoding the polypeptide of interest, although certain secretory signal sequences may be positioned elsewhere in the nucleic acid sequence of interest (see, e.g., Welch et al., U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat. No. 5,143,830). In a preferred embodiment the signal peptide comprises the amino acid sequence SEQ ID NO: 1 or 2.

Those skilled in the art will recognize that, in view of the degeneracy of the genetic code, considerable sequence variation is possible among these polynucleotide molecules. Preferably, the nucleic acid sequences of the present invention are codon-optimized for expression in mammalian cells, preferably for expression in human cells. Codon-optimization refers to the exchange in a sequence of interest of codons that are generally rare in highly expressed genes of a given species by codons that are generally frequent in highly expressed genes of such species, such codons encoding the amino acids as the codons that are being exchanged.

Methods of Engineering Immune Cells Endowed with CARs:

The present invention encompasses the method of preparing immune cells for immunotherapy comprising introducing ex-vivo into said immune cells the polynucleotides or vectors encoding one of the ROR1 CAR as previously described.

In a preferred embodiment, said polynucleotides are included in lentiviral vectors in view of being stably expressed in the immune cells.

According to further embodiments, said method further comprises the step of genetically modifying said cell to make them more suitable for allogeneic transplantation.

According to a first aspect, the immune cell can be made allogeneic, for instance, by inactivating at least one gene expressing one or more component of T-cell receptor (TCR) as described in WO 2013/176915, which can be combined with the inactivation of a gene encoding or regulating HLA or β2m protein expression. Accordingly the risk of graft versus host syndrome and graft rejection is significantly reduced.

According to another aspect, the immune cells can be further genetically engineered to improve their resistance to immunosuppressive drugs or chemotherapy treatments, which are used as standard care for treating ROR1 positive malignant cells. For instance, CD52 and glucocorticoid receptors (GR), which are drug targets of Campath (alemtuzumab) and glucocorticoids treatments, can be inactivated to make the cells resistant to these treatments and give them a competitive advantage over patient's own T-cells not endowed with specific ROR1 CARs. Expression of CD3 gene can also be suppressed or reduced to confer resistance to Teplizumab, which is another immune suppressive drug. Expression of HPRT can also be suppressed or reduced according to the invention to confer resistance to 6-thioguanine, a cytostatic agent commonly used in chemotherapy especially for the treatment of acute lymphoblastic leukemia.

According to further aspect of the invention, the immune cells can be further manipulated to make them more active or limit exhaustion, by inactivating genes encoding proteins that act as “immune checkpoints” that act as regulators of T-cells activation, such as PDCD1 or CTLA-4. Examples of genes, which expression could be reduced or suppressed are indicated in Table 9.

TABLE 9 List of genes encoding immune checkpoint proteins. Genes that can be inactivated Pathway In the pathway Co-inhibitory CTLA4 (CD152) CTLA4, PPP2CA, PPP2CB, PTPN6, receptors PTPN22 PDCD1 (PD-1, CD279) PDCD1 CD 223 (lag3) LAG3 HAVCR2 (tim3) HAVCR2 BTLA(cd272) BTLA CD160(by55) CD160 IgSF family TIGIT CD96 CRTAM LAIR1(cd305) LAIR1 SIGLECs SIGLEC7 SIGLEC9 CD244(2b4) CD244 Death receptors TRAIL INFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7 FAS FADD, FAS Cytokine signalling TGF-beta signaling TGFBRII, TGFBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1 IL10 signalling IL10RA, IL10RB, HMOX2 IL6 signalling IL6R, IL6ST Prevention of TCR CSK, PAG1 signalling SIT1 Induced Treg induced Treg FOXP3 Transcription transcription factors PRDM1 (=blimp1, heterozygotes mice factors controlling controlling exhaustion control chronic viral infection better exhaustion than wt or conditional KO) BATF Hypoxia mediated iNOS induced guanylated GUCY1A2, GUCY1A3, GUCY1B2, tolerance cyclase GUCY1B3

In a preferred embodiment said method of further engineering the immune cells involves introducing into said T cells polynucleotides, in particular mRNAs, encoding specific rare-cutting endonuclease to selectively inactivate the genes, as those mentioned above, by DNA cleavage. In a more preferred embodiment said rare-cutting endonucleases are TALE-nucleases or Cas9 endonuclease. TAL-nucleases have so far proven higher specificity and cleavage efficiency over the other types of rare-cutting endonucleases, making them the endonucleases of choice for producing of the engineered immune cells on a large scale with a constant turn-over.

Delivery Methods

The different methods described above involve introducing CAR into a cell. As non-limiting example, said CAR can be introduced as transgenes encoded by one plasmid vector. Said plasmid vector can also contain a selection marker which provides for identification and/or selection of cells which received said vector.

Polypeptides may be synthesized in situ in the cell as a result of the introduction of polynucleotides encoding said polypeptides into the cell. Alternatively, said polypeptides could be produced outside the cell and then introduced thereto. Methods for introducing a polynucleotide construct into cells are known in the art and including as non-limiting examples stable transformation methods wherein the polynucleotide construct is integrated into the genome of the cell, transient transformation methods wherein the polynucleotide construct is not integrated into the genome of the cell and virus mediated methods. Said polynucleotides may be introduced into a cell by for example, recombinant viral vectors (e.g. retroviruses, adenoviruses), liposome and the like. For example, transient transformation methods include for example microinjection, electroporation or particle bombardment. Said polynucleotides may be included in vectors, more particularly plasmids or virus, in view of being expressed in cells.

Engineered Immune Cells

The present invention also relates to isolated cells or cell lines susceptible to be obtained by said method to engineer cells. In particular said isolated cell comprises at least one CAR as described above. In another embodiment, said isolated cell comprises a population of CARs each one comprising different extracellular ligand binding domains. In particular, said isolated cell comprises exogenous polynucleotide sequence encoding CAR. Genetically modified immune cells of the present invention are activated and proliferate independently of antigen binding mechanisms.

In the scope of the present invention is also encompassed an isolated immune cell, preferably a T-cell obtained according to any one of the methods previously described. Said immune cell refers to a cell of hematopoietic origin functionally involved in the initiation and/or execution of innate and/or adaptive immune response. Said immune cell according to the present invention can be derived from a stem cell. The stem cells can be adult stem cells, non-human embryonic stem cells, more particularly non-human stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells or hematopoietic stem cells. Representative human cells are CD34+ cells. Said isolated cell can also be a dendritic cell, killer dendritic cell, a mast cell, a NK-cell, a B-cell or a T-cell selected from the group consisting of inflammatory T-lymphocytes, cytotoxic T-lymphocytes, regulatory T-lymphocytes or helper T-lymphocytes. In another embodiment, said cell can be derived from the group consisting of CD4+T-lymphocytes and CD8+T-lymphocytes. Prior to expansion and genetic modification of the cells of the invention, a source of cells can be obtained from a subject through a variety of non-limiting methods. Cells can be obtained from a number of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the present invention, any number of T cell lines available and known to those skilled in the art, may be used. In another embodiment, said cell can be derived from a healthy donor, from a patient diagnosed with cancer or from a patient diagnosed with an infection. In another embodiment, said cell is part of a mixed population of cells which present different phenotypic characteristics. In the scope of the present invention is also encompassed a cell line obtained from a transformed T-cell according to the method previously described. Modified cells resistant to an immunosuppressive treatment and susceptible to be obtained by the previous method are encompassed in the scope of the present invention.

As a preferred embodiment, the present invention provides T-cells or a population of T-cells endowed with a ROR1 CAR as described above, that do not express functional TCR and that a reactive towards ROR1 positive cells, for their allogeneic transplantation into patients.

Activation and Expansion of T Cells

Whether prior to or after genetic modification of the T cells, even if the genetically modified immune cells of the present invention are activated and proliferate independently of antigen binding mechanisms, the immune cells, particularly T-cells of the present invention can be further activated and expanded generally using methods as described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005. T cells can be expanded in vitro or in vivo.

Generally, the T cells of the invention are expanded by contact with an agent that stimulates a CD3 TCR complex and a co-stimulatory molecule on the surface of the T cells to create an activation signal for the T-cell. For example, chemicals such as calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA), or mitogenic lectins like phytohaemagglutinin (PHA) can be used to create an activation signal for the T-cell.

As non-limiting examples, T cell populations may be stimulated in vitro such as by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For co-stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells. Conditions appropriate for T cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 5, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-g, 1L-4, 1L-7, GM-CSF, -10, -2, 1L-15, TGFp, and TNF- or any other additives for the growth of cells known to the skilled artisan. Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanoi. Media can include RPMI 1640, A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo 1, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and/or an amount of cytokine(s) sufficient for the growth and expansion of T cells. Antibiotics, e.g., penicillin and streptomycin, are included only in experimental cultures, not in cultures of cells that are to be infused into a subject. The target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air plus 5% CO2). T cells that have been exposed to varied stimulation times may exhibit different characteristics.

In another particular embodiment, said cells can be expanded by co-culturing with tissue or cells. Said cells can also be expanded in vivo, for example in the subject's blood after administrating said cell into the subject.

Therapeutic Applications

In another embodiment, isolated cell obtained by the different methods or cell line derived from said isolated cell as previously described can be used as a medicament. In another embodiment, said medicament can be used for treating cancer, particularly for the treatment of B-cell lymphomas and leukemia in a patient in need thereof. In another embodiment, said isolated cell according to the invention or cell line derived from said isolated cell can be used in the manufacture of a medicament for treatment of a cancer in a patient in need thereof.

In another aspect, the present invention relies on methods for treating patients in need thereof, said method comprising at least one of the following steps:

-   -   (a) providing an immune-cell obtainable by any one of the         methods previously described;     -   (b) Administrating said transformed immune cells to said         patient,

On one embodiment, said T cells of the invention can undergo robust in vivo T cell expansion and can persist for an extended amount of time.

Said treatment can be ameliorating, curative or prophylactic. It may be either part of an autologous immunotherapy or part of an allogenic immunotherapy treatment. By autologous, it is meant that cells, cell line or population of cells used for treating patients are originating from said patient or from a Human Leucocyte Antigen (HLA) compatible donor. By allogeneic is meant that the cells or population of cells used for treating patients are not originating from said patient but from a donor.

Cells that can be used with the disclosed methods are described in the previous section. Said treatment can be used to treat patients diagnosed wherein a pre-malignant or malignant cancer condition characterized by ROR1-expressing cells, especially by an overabundance of ROR1-expressing cells. Such conditions are found in hematologic cancers, such as leukemia or malignant lymphoproliferative disorders.

Leukemia can be acute myelogenous leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, acute lymphoid leukemia, chronic lymphoid leukemia, and myelodysplastic syndrome.

Lymphoproliferative disorder can be lymphoma, in particular chronic lymphocytic leukemia, non-Hodgkin's lymphoma, Burkitt's lymphoma, and follicular lymphoma (small cell and large cell).

According to one preferred embodiment, said engineered T cells are provided for the treatment of the Chronic Lymphocytic Leukemia (CLL) or the Small Lymphocytic Lymphoma (SLL).

According to another preferred embodiment, said treatment of CLL or SLL is administered to patients who have been lympho-depleted before the ROR1-CAR-T cell infusion. Said lympho-depletion is performed usually by chemotherapy, and preferably by using drugs as fludarabine (F), cyclophosphamide (C), bendamustine (B) or rituximab (R) or a combination thereof. Typically, the combination of ER or FBR can be used for lympho-depletion prior to CAR-T cell administration.

According to another preferred embodiment, said engineered T cells are provided for the treatment of Mantle Cell Lymphoma (MCL, Acute Lymphoblastic Leukemia (ALL) with a t(1;19) chromosome translocation.

Cancers that may be treated may comprise nonsolid tumors (such as hematological tumors, including but not limited to pre-B ALL (pedriatic indication), adult ALL, mantle cell lymphoma, diffuse large B-cell lymphoma and the like. Types of cancers to be treated with the CARs of the invention include, but are not limited leukemia or lymphoid malignancies. Adult tumors/cancers and pediatric tumors/cancers are also included.

Also, solid tumors such as breast, colon, lung, and kidney tumors can be treated by the CARs of the invention. Also, the engineered T cells of the invention can be used as a treatment of pancreas, renal or ovarian cancers.

The treatment with the engineered immune cells according to the invention may be in combination with one or more therapies against cancer selected from the group of antibodies therapy, chemotherapy, cytokines therapy, dendritic cell therapy, gene therapy, hormone therapy, laser light therapy and radiation therapy.

According to a preferred embodiment of the invention, said treatment can be administrated into patients undergoing an immunosuppressive treatment. Indeed, the present invention preferably relies on cells or population of cells, which have been made resistant to at least one immunosuppressive agent due to the inactivation of a gene encoding a receptor for such immunosuppressive agent. In this aspect, the immunosuppressive treatment should help the selection and expansion of the T-cells according to the invention within the patient.

The administration of the cells or population of cells according to the present invention may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous or intralymphatic injection, or intraperitoneally. In one embodiment, the cell compositions of the present invention are preferably administered by intravenous injection.

The administration of the cells or population of cells can consist of the administration of 10⁴-10⁹ cells per kg body weight, preferably 10⁵ to 10⁶ cells/kg body weight including all integer values of cell numbers within those ranges. The cells or population of cells can be administrated in one or more doses. In another embodiment, said effective amount of cells are administrated as a single dose. In another embodiment, said effective amount of cells are administrated as more than one dose over a period time. Timing of administration is within the judgment of managing physician and depends on the clinical condition of the patient. The cells or population of cells may be obtained from any source, such as a blood bank or a donor. While individual needs vary, determination of optimal ranges of effective amounts of a given cell type for a particular disease or conditions within the skill of the art. An effective amount means an amount which provides a therapeutic or prophylactic benefit. The dosage administrated will be dependent upon the age, health and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment and the nature of the effect desired.

In another embodiment, said effective amount of cells or composition comprising those cells are administrated parenterally. Said administration can be an intravenous administration. Said administration can be directly done by injection within a tumor.

In certain embodiments of the present invention, cells are administered to a patient in conjunction with (e.g., before, simultaneously or following) any number of relevant treatment modalities, including but not limited to treatment with agents such as antiviral therapy, cidofovir and interleukin-2, Cytarabine (also known as ARA-C) or nataliziimab treatment for MS patients or efaliztimab treatment for psoriasis patients or other treatments for PML patients. In further embodiments, the T cells of the invention may be used in combination with chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAM PATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludaribine, cyclosporin, FK506, rapamycin, mycoplienolic acid, steroids, FR901228, cytokines, and irradiation. These drugs inhibit either the calcium dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase that is important for growth factor induced signaling (rapamycin) (Henderson, Naya et al. 1991; Liu, Albers et al. 1992; Bierer, Hollander et al. 1993). In a further embodiment, the cell compositions of the present invention are administered to a patient in conjunction with (e.g., before, simultaneously or following) bone marrow transplantation, T cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH, In another embodiment, the cell compositions of the present invention are administered following B-cell ablative therapy such as agents that react with CD20, e.g., Rituxan. For example, in one embodiment, subjects may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, following the transplant, subjects receive an infusion of the expanded immune cells of the present invention. In an additional embodiment, expanded cells are administered before or following surgery.

OTHER DEFINITIONS

-   -   Unless otherwise specified, “a,” “an,” “the,” and “at least one”         are used interchangeably and mean one or more than one. Amino         acid residues in a polypeptide sequence are designated herein         according to the one-letter code, in which, for example, Q means         Gln or Glutamine residue, R means Arg or Arginine residue and D         means Asp or Aspartic acid residue.     -   Amino acid substitution means the replacement of one amino acid         residue with another, for instance the replacement of an         Arginine residue with a Glutamine residue in a peptide sequence         is an amino acid substitution.     -   Nucleotides are designated as follows: one-letter code is used         for designating the base of a nucleoside: a is adenine, t is         thymine, c is cytosine, and g is guanine. For the degenerated         nucleotides, r represents g or a (purine nucleotides), k         represents g or t, s represents g or c, w represents a or t, m         represents a or c, y represents t or c (pyrimidine nucleotides),         d represents g, a or t, v represents g, a or c, b represents g,         t or c, h represents a, t or c, and n represents g, a, t or c.     -   “As used herein, “nucleic acid” or “polynucleotides” refers to         nucleotides and/or polynucleotides, such as deoxyribonucleic         acid (DNA) or ribonucleic acid (RNA), oligonucleotides,         fragments generated by the polymerase chain reaction (PCR), and         fragments generated by any of ligation, scission, endonuclease         action, and exonuclease action. Nucleic acid molecules can be         composed of monomers that are naturally-occurring nucleotides         (such as DNA and RNA), or analogs of naturally-occurring         nucleotides (e.g., enantiomeric forms of naturally-occurring         nucleotides), or a combination of both. Modified nucleotides can         have alterations in sugar moieties and/or in pyrimidine or         purine base moieties. Sugar modifications include, for example,         replacement of one or more hydroxyl groups with halogens, alkyl         groups, amines, and azido groups, or sugars can be         functionalized as ethers or esters. Moreover, the entire sugar         moiety can be replaced with sterically and electronically         similar structures, such as aza-sugars and carbocyclic sugar         analogs. Examples of modifications in a base moiety include         alkylated purines and pyrimidines, acylated purines or         pyrimidines, or other well-known heterocyclic substitutes.         Nucleic acid monomers can be linked by phosphodiester bonds or         analogs of such linkages. Nucleic acids can be either single         stranded or double stranded.     -   By chimeric antigen receptor (CAR) is intended molecules that         combine a binding domain against a component present on the         target cell, for example an antibody-based specificity for a         desired antigen (e.g., tumor antigen) with a T cell         receptor-activating intracellular domain to generate a chimeric         protein that exhibits a specific anti-target cellular immune         activity. Generally, CAR consists of an extracellular single         chain antibody (scFvFc) fused to the intracellular signaling         domain of the T cell antigen receptor complex zeta chain         (scFvFc:ζ) and have the ability, when expressed in T cells, to         redirect antigen recognition based on the monoclonal antibody's         specificity. One example of CAR used in the present invention is         a CAR directing against ROR1 antigen and can comprise as         non-limiting example the amino acid sequences: SEQ ID NO: 79 to         138.     -   The term “endonuclease” refers to any wild-type or variant         enzyme capable of catalyzing the hydrolysis (cleavage) of bonds         between nucleic acids within a DNA or RNA molecule, preferably a         DNA molecule. Endonucleases do not cleave the DNA or RNA         molecule irrespective of its sequence, but recognize and cleave         the DNA or RNA molecule at specific polynucleotide sequences,         further referred to as “target sequences” or “target sites”.         Endonucleases can be classified as rare-cutting endonucleases         when having typically a polynucleotide recognition site greater         than 12 base pairs (bp) in length, more preferably of 14-55 bp.         Rare-cutting endonucleases significantly increase HR by inducing         DNA double-strand breaks (DSBs) at a defined locus (Perrin,         Buckle et al. 1993; Rouet, Smih et al. 1994; Choulika, Perrin et         al. 1995; Pingoud and Silva 2007). Rare-cutting endonucleases         can for example be a homing endonuclease (Paques and Duchateau         2007), a chimeric Zinc-Finger nuclease (ZFN) resulting from the         fusion of engineered zinc-finger domains with the catalytic         domain of a restriction enzyme such as FokI (Porteus and Carroll         2005), a Cas9 endonuclease from CRISPR system (Gasiunas,         Barrangou et al. 2012; Jinek, Chylinski et al. 2012; Cong, Ran         et al. 2013; Mali, Yang et al. 2013) or a chemical endonuclease         (Eisenschmidt, Lanio et al. 2005; Arimondo, Thomas et al. 2006).         In chemical endonucleases, a chemical or peptidic cleaver is         conjugated either to a polymer of nucleic acids or to another         DNA recognizing a specific target sequence, thereby targeting         the cleavage activity to a specific sequence. Chemical         endonucleases also encompass synthetic nucleases like conjugates         of orthophenanthroline, a DNA cleaving molecule, and         triplex-forming oligonucleotides (TFOs), known to bind specific         DNA sequences (Kalish and Glazer 2005). Such chemical         endonucleases are comprised in the term “endonuclease” according         to the present invention.     -   By a “TALE-nuclease” (TALEN) is intended a fusion protein         consisting of a nucleic acid-binding domain typically derived         from a Transcription Activator Like Effector (TALE) and one         nuclease catalytic domain to cleave a nucleic acid target         sequence. The catalytic domain is preferably a nuclease domain         and more preferably a domain having endonuclease activity, like         for instance I-TevI, ColE7, NucA and Fok-I. In a particular         embodiment, the TALE domain can be fused to a meganuclease like         for instance I-CreI and 1-OnuI or functional variant thereof. In         a more preferred embodiment, said nuclease is a monomeric         TALE-Nuclease. A monomeric TALE-Nuclease is a TALE-Nuclease that         does not require dimerization for specific recognition and         cleavage, such as the fusions of engineered TAL repeats with the         catalytic domain of I-TevI described in WO2012138927.         Transcription Activator like Effector (TALE) are proteins from         the bacterial species Xanthomonas comprise a plurality of         repeated sequences, each repeat comprising di-residues in         position 12 and 13 (RVD) that are specific to each nucleotide         base of the nucleic acid targeted sequence. Binding domains with         similar modular base-per-base nucleic acid binding properties         (MBBBD) can also be derived from new modular proteins recently         discovered by the applicant in a different bacterial species.         The new modular proteins have the advantage of displaying more         sequence variability than TAL repeats. Preferably, RVDs         associated with recognition of the different nucleotides are HD         for recognizing C, NG for recognizing T, NI for recognizing A,         NN for recognizing G or A, NS for recognizing A, C, G or T, HG         for recognizing T, IG for recognizing T, NK for recognizing G,         HA for recognizing C, ND for recognizing C, HI for recognizing         C, HN for recognizing G, NA for recognizing G, SN for         recognizing G or A and YG for recognizing T, TL for recognizing         A, VT for recognizing A or G and SW for recognizing A. In         another embodiment, critical amino acids 12 and 13 can be         mutated towards other amino acid residues in order to modulate         their specificity towards nucleotides A, T, C and G and in         particular to enhance this specificity. TALE-nuclease have been         already described and used to stimulate gene targeting and gene         modifications (Boch, Scholze et al. 2009; Moscou and Bogdanove         2009; Christian, Cermak et al. 2010; Li, Huang et al. 2011).         Custom-made TAL-nucleases are commercially available under the         trade name TALEN™ (Cellectis, 8 rue de la Croix Jarry, 75013         Paris, France).

The rare-cutting endonuclease according to the present invention can also be a Cas9 endonuclease. Recently, a new genome engineering tool has been developed based on the RNA-guided Cas9 nuclease (Gasiunas, Barrangou et al. 2012; Jinek, Chylinski et al. 2012; Cong, Ran et al. 2013; Mali, Yang et al. 2013) from the type II prokaryotic CRISPR (Clustered Regularly Interspaced Short palindromic Repeats) adaptive immune system (see for review (Sorek, Lawrence et al. 2013)). The CRISPR Associated (Cas) system was first discovered in bacteria and functions as a defense against foreign DNA, either viral or plasmid. CRISPR-mediated genome engineering first proceeds by the selection of target sequence often flanked by a short sequence motif, referred as the protospacer adjacent motif (PAM). Following target sequence selection, a specific crRNA, complementary to this target sequence is engineered. Trans-activating crRNA (tracrRNA) required in the CRISPR type II systems paired to the crRNA and bound to the provided Cas9 protein. Cas9 acts as a molecular anchor facilitating the base pairing of tracRNA with cRNA (Deltcheva, Chylinski et al. 2011). In this ternary complex, the dual tracrRNA:crRNA structure acts as guide RNA that directs the endonuclease Cas9 to the cognate target sequence. Target recognition by the Cas9-tracrRNA:crRNA complex is initiated by scanning the target sequence for homology between the target sequence and the crRNA. In addition to the target sequence-crRNA complementarity, DNA targeting requires the presence of a short motif adjacent to the protospacer (protospacer adjacent motif—PAM). Following pairing between the dual-RNA and the target sequence, Cas9 subsequently introduces a blunt double strand break 3 bases upstream of the PAM motif (Garneau, Dupuis et al. 2010).

Rare-cutting endonuclease can be a homing endonuclease, also known under the name of meganuclease. Such homing endonucleases are well-known to the art (Stoddard 2005). Homing endonucleases recognize a DNA target sequence and generate a single- or double-strand break. Homing endonucleases are highly specific, recognizing DNA target sites ranging from 12 to 45 base pairs (bp) in length, usually ranging from 14 to 40 bp in length. The homing endonuclease according to the invention may for example correspond to a LAGLIDADG endonuclease, to a HNH endonuclease, or to a GIY-YIG endonuclease. Preferred homing endonuclease according to the present invention can be an I-CreI variant.

-   -   By “delivery vector” or “delivery vectors” is intended any         delivery vector which can be used in the present invention to         put into cell contact (i.e “contacting”) or deliver inside cells         or subcellular compartments (i.e “introducing”) agents/chemicals         and molecules (proteins or nucleic acids) needed in the present         invention. It includes, but is not limited to liposomal delivery         vectors, viral delivery vectors, drug delivery vectors, chemical         carriers, polymeric carriers, lipoplexes, polyplexes,         dendrimers, microbubbles (ultrasound contrast agents),         nanoparticles, emulsions or other appropriate transfer vectors.         These delivery vectors allow delivery of molecules, chemicals,         macromolecules (genes, proteins), or other vectors such as         plasmids, peptides developed by Diatos. In these cases, delivery         vectors are molecule carriers. By “delivery vector” or “delivery         vectors” is also intended delivery methods to perform         transfection.     -   The terms “vector” or “vectors” refer to a nucleic acid molecule         capable of transporting another nucleic acid to which it has         been linked. A “vector” in the present invention includes, but         is not limited to, a viral vector, a plasmid, a RNA vector or a         linear or circular DNA or RNA molecule which may consists of a         chromosomal, non-chromosomal, semi-synthetic or synthetic         nucleic acids. Preferred vectors are those capable of autonomous         replication (episomal vector) and/or expression of nucleic acids         to which they are linked (expression vectors). Large numbers of         suitable vectors are known to those of skill in the art and         commercially available.

Viral vectors include retrovirus, adenovirus, parvovirus (e. g. adenoassociated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e. g., influenza virus), rhabdovirus (e. g., rabies and vesicular stomatitis virus), paramyxovirus (e. g. measles and Sendai), positive strand RNA viruses such as picornavirus and alphavirus, and double-stranded DNA viruses including adenovirus, herpesvirus (e. g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e. g., vaccinia, fowlpox and canarypox). Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example. Examples of retroviruses include: avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).

-   -   By “lentiviral vector” is meant HIV-Based lentiviral vectors         that are very promising for gene delivery because of their         relatively large packaging capacity, reduced immunogenicity and         their ability to stably transduce with high efficiency a large         range of different cell types. Lentiviral vectors are usually         generated following transient transfection of three (packaging,         envelope and transfer) or more plasmids into producer cells.         Like HIV, lentiviral vectors enter the target cell through the         interaction of viral surface glycoproteins with receptors on the         cell surface. On entry, the viral RNA undergoes reverse         transcription, which is mediated by the viral reverse         transcriptase complex. The product of reverse transcription is a         double-stranded linear viral DNA, which is the substrate for         viral integration in the DNA of infected cells. By “integrative         lentiviral vectors (or LV)”, is meant such vectors as         nonlimiting example, that are able to integrate the genome of a         target cell. At the opposite by “non-integrative lentiviral         vectors (or NILV)” is meant efficient gene delivery vectors that         do not integrate the genome of a target cell through the action         of the virus integrase.     -   Delivery vectors and vectors can be associated or combined with         any cellular permeabilization techniques such as sonoporation or         electroporation or derivatives of these techniques.     -   By cell or cells is intended any eukaryotic living cells,         primary cells and cell lines derived from these organisms for in         vitro cultures.     -   By “primary cell” or “primary cells” are intended cells taken         directly from living tissue (i.e. biopsy material) and         established for growth in vitro, that have undergone very few         population doublings and are therefore more representative of         the main functional components and characteristics of tissues         from which they are derived from, in comparison to continuous         tumorigenic or artificially immortalized cell lines.

As non-limiting examples cell lines can be selected from the group consisting of CHO-K1 cells; HEK293 cells; Caco2 cells; U2-OS cells; NIH 3T3 cells; NSO cells; SP2 cells; CHO-S cells; DG44 cells; K-562 cells, U-937 cells; MRC5 cells; IMR90 cells; Jurkat cells; HepG2 cells; HeLa cells; HT-1080 cells; HCT-116 cells; Hu-h7 cells; Huvec cells; Molt 4 cells.

All these cell lines can be modified by the method of the present invention to provide cell line models to produce, express, quantify, detect, study a gene or a protein of interest; these models can also be used to screen biologically active molecules of interest in research and production and various fields such as chemical, biofuels, therapeutics and agronomy as non-limiting examples.

-   -   by “mutation” is intended the substitution, deletion, insertion         of up to one, two, three, four, five, six, seven, eight, nine,         ten, eleven, twelve, thirteen, fourteen, fifteen, twenty, twenty         five, thirty, forty, fifty, or more nucleotides/amino acids in a         polynucleotide (cDNA, gene) or a polypeptide sequence. The         mutation can affect the coding sequence of a gene or its         regulatory sequence. It may also affect the structure of the         genomic sequence or the structure/stability of the encoded mRNA.     -   by “variant(s)”, it is intended a repeat variant, a variant, a         DNA binding variant, a TALE-nuclease variant, a polypeptide         variant obtained by mutation or replacement of at least one         residue in the amino acid sequence of the parent molecule.     -   by “functional variant” is intended a catalytically active         mutant of a protein or a protein domain; such mutant may have         the same activity compared to its parent protein or protein         domain or additional properties, or higher or lower activity.     -   “identity” refers to sequence identity between two nucleic acid         molecules or polypeptides. Identity can be determined by         comparing a position in each sequence which may be aligned for         purposes of comparison. When a position in the compared sequence         is occupied by the same base, then the molecules are identical         at that position. A degree of similarity or identity between         nucleic acid or amino acid sequences is a function of the number         of identical or matching nucleotides at positions shared by the         nucleic acid sequences. Various alignment algorithms and/or         programs may be used to calculate the identity between two         sequences, including FASTA, or BLAST which are available as a         part of the GCG sequence analysis package (University of         Wisconsin, Madison, Wis.), and can be used with, e.g., default         setting. For example, polypeptides having at least 70%, 85%,         90%, 95%, 98% or 99% identity to specific polypeptides described         herein and preferably exhibiting substantially the same         functions, as well as polynucleotide encoding such polypeptides,         are contemplated. Unless otherwise indicated a similarity score         will be based on use of BLOSUM62. When BLASTP is used, the         percent similarity is based on the BLASTP positives score and         the percent sequence identity is based on the BLASTP identities         score. BLASTP “Identities” shows the number and fraction of         total residues in the high scoring sequence pairs which are         identical; and BLASTP “Positives” shows the number and fraction         of residues for which the alignment scores have positive values         and which are similar to each other. Amino acid sequences having         these degrees of identity or similarity or any intermediate         degree of identity of similarity to the amino acid sequences         disclosed herein are contemplated and encompassed by this         disclosure. The polynucleotide sequences of similar polypeptides         are deduced using the genetic code and may be obtained by         conventional means, in particular by reverse translating its         amino acid sequence using the genetic code.     -   “signal-transducing domain” or “co-stimulatory ligand” refers to         a molecule on an antigen presenting cell that specifically binds         a cognate co-stimulatory molecule on a T-cell, thereby providing         a signal which, in addition to the primary signal provided by,         for instance, binding of a TCR/CD3 complex with an MHC molecule         loaded with peptide, mediates a T cell response, including, but         not limited to, proliferation activation, differentiation and         the like. A co-stimulatory ligand can include but is not limited         to CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L,         inducible costimulatory igand (ICOS-L), intercellular adhesion         molecule (ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB,         HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, an agonist         or antibody that binds Toll ligand receptor and a ligand that         specifically binds with B7-H3. A co-stimulatory ligand also         encompasses, inter alia, an antibody that specifically binds         with a co-stimulatory molecule present on a T cell, such as but         not limited to, CD27, CD28, 4-IBB, OX40, CD30, CD40, PD-1, ICOS,         lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7,         LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83.

A “co-stimulatory molecule” refers to the cognate binding partner on a Tcell that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the cell, such as, but not limited to proliferation. Co-stimulatory molecules include, but are not limited to an MHC class I molecule, BILA and Toll ligand receptor.

A “co-stimulatory signal” as used herein refers to a signal, which in combination with primary signal, such as TCR/CD3 ligation, leads to T cell proliferation and/or upregulation or downregulation of key molecules.

The term “extracellular ligand-binding domain” as used herein is defined as an oligo- or polypeptide that is capable of binding a ligand. Preferably, the domain will be capable of interacting with a cell surface molecule. For example, the extracellular ligand-binding domain may be chosen to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state. Thus examples of cell surface markers that may act as ligands include those associated with viral, bacterial and parasitic infections, autoimmune disease and cancer cells.

The term “subject” or “patient” as used herein includes all members of the animal kingdom including non-human primates and humans.

The above written description of the invention provides a manner and process of making and using it such that any person skilled in this art is enabled to make and use the same, this enablement being provided in particular for the subject matter of the appended claims, which make up a part of the original description.

Where a numerical limit or range is stated herein, the endpoints are included. Also, all values and subranges within a numerical limit or range are specifically included as if explicitly written out.

The above description is presented to enable a person skilled in the art to make and use the invention, and is provided in the context of a particular application and its requirements. Various modifications to the preferred embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of the invention. Thus, this invention is not intended to be limited to the embodiments shown, but is to be accorded the widest scope consistent with the principles and features disclosed herein.

Having generally described this invention, a further understanding can be obtained by reference to certain specific examples, which are provided herein for purposes of illustration only, and are not intended to be limiting unless otherwise specified.

General Method

Primary Cells Peripheral blood mononuclear cells were isolated by density gradient centrifugation from buffy coats from healthy volunteer donors (Etablissement Français du Sang). T lymphocytes were then purified using the EasySep human T cell enrichment kit (Stemcell Technologies), and activated with Dynabeads Human T-Activator CD3/CD28 (Life Technologies) in X-vivo 15 medium (Lonza) supplemented with 20 ng/ml IL-2 (Miltenyi) and 5% human AB serum (Seralab).

Cell Lines

The K562, Jeko-1, MDA-MB-231, PC-3 and MCF-7 cell lines were obtained from the American Type Culture Collection. K562 cells were cultured in IMDM supplemented with 10% heat-inactivated FCS, 2 mmol/L L-glutamine and 100 units/ml penicillin, and 100 μg/mL streptomycin. Jeko-1 cells were cultured in RPMI 1640 supplemented with 20% heat-inactivated FCS, 2 mmol/L L-glutamine and 100 units/ml penicillin, and 100m/mL streptomycin. MDA-MB-231 cells were cultured in DMEM supplemented with 10% heat-inactivated FCS, 2 mmol/L L-glutamine and 100 units/ml penicillin, and 100 μg/mL streptomycin. PC-3 cells were cultured in F-12K supplemented with 10% heat-inactivated FCS, 2 mmol/L L-glutamine and 100 units/ml penicillin, and 100 μg/mL streptomycin. MCF-7 cells were cultured in DMEM supplemented with 10% heat-inactivated FCS, 2 mmol/L L-glutamine and 100 units/ml penicillin, and 100m/mL streptomycin and 0.01 mg/ml human insulin.

Quantification of ROR1 Cell Surface Expression

The number of ROR1 surface molecules on different human cell lines was determined by saturation binding using the monoclonal anti-ROR1 antibody clone 2A (Miltenyi) and the Dako QiFIKIT according to the manufacturer's instructions.

Synthesis of DNA Encoding scCARs

The DNA encoding the scCARs was synthesized by GenScript.

Construction of In Vitro Transcription mRNA Vectors for scCARs

The DNA encoding the scCARs was cloned in the plasmid pCLS9632 between the T7 promoter and the BGH poly A.

RNA In Vitro Transcription

mRNA encoding the scCAR were in vitro transcribed and polyadenylated using the mMessage mMachine T7 Ultra kit (Life technologies) following the manufacturer's instructions. RNAs were purified with RNeasy columns (Qiagen), eluted in cytoporation medium T (Harvard Apparatus), and quantified by measuring absorbance at 260 nm using a Nanodrop ND-1000 spectrophotometer. Quality of the RNA was verified on a denaturing formaldehyde/MOPS agarose gel.

RNA Electroporation of T Cells

4-5 days or 11-12 post-activation, T lymphocytes were transfected by electrotransfer of messenger RNA using an AgilePulse MAX system (Harvard Apparatus). I. 5×10⁶cells were mixed with 15 μg of the mRNA encoding the scCAR into a 0.4 cm cuvette. The electroporation was performed according to the protocol set forth in WO2013176915. Following electroporation, cells were diluted into culture medium and incubated at 37° C./5% CO₂.

Detection of scCAR

By flow cytometry: The T cells were stained with PE-labeled polyclonal goat anti-mouse (Fab)₂ antibodies (Jackson Immunoresearch) or biotin-labeled protein L (GenScript) followed by phycoerythrin-labeled streptavidin (BD pharmingen), and finally analysed using the MACSQuant flow cytometer (Miltenyi).

By Western blotting: 1×10⁶ T cells were lysed in 50 μl RIPA buffer containing 1 mM orthovanadate, 3 μg/ml of protease inhibitor and 2 mM of PMSF. Cells lysates were separated by SDS-PAGE on a Any kD™ acrylamide gel (BioRad). After transfer to a nitrocellulose membrane, this was incubated with a mouse anti-human CD3z (pharmingen) and then with a goat anti-mouse IgG horseradish peroxidase-conjugated antibody (Sigma). Antibody binding was revealed by using the ECL kit (Pierce).

Degranulation Assay

5×10⁴ T cells were co-cultured with 5×10⁴ ROR1-positive or ROR1-negative cells in 0.1 ml per well in a 96-well plate. APC-labeled anti-CD107a (BD Biosciences) was added at the beginning of the co-culture in addition to 1 μg/ml of anti-CD49d (BD Biosciences), 1 μg/ml of anti-CD28 (Miltenyi), and 1× Monensin solution (eBioscience). After a 6h incubation, the cells were stained with a fixable viability dye (eBioscience) and vioblue-labeled anti-CD8 (Miltenyi) and analyzed using the MACSQuant flow cytometer (Miltenyi). Degranulating cytotoxic T cells correspond to CD8+CD107a+ cells.

Cytokine Release Assay

5×10⁴ T cells were co-cultured with 5×10⁴ ROR1-positive or ROR1-negative cells in 0.1 ml per well in a 96-well plate. After a 24 hours incubation, the culture supernatants were collected and analysed for IFNγROR1-positive or ROR1-negative cells.

Cytotoxicity Assay

With Adherent Target Cells:

2×10⁴ ROR1-positive or ROR1-negative cells were seeded in 0.1 ml per well in a 96 well plate. The day after the plating, the ROR1-positive and the ROR1-negative cells were labeled with CellTrace CFSE and co-cultured with 4×10⁵ T cells for 4 hours. The cells were then harvested, stained with a fixable viability dye (eBioscience) and analyzed using the MACSQuant flow cytometer (Miltenyi).

The percentage of specific lysis was calculated using the following formula:

${{\%\mspace{14mu}{cell}\mspace{14mu}{lysis}} = {{100\%} - \frac{\frac{\%\mspace{14mu}{viable}\mspace{14mu}{target}\mspace{14mu}{cells}\mspace{14mu}{upon}\mspace{14mu}{coculture}\mspace{11mu}{with}\mspace{14mu}{CAR}\mspace{14mu}{modified}\mspace{14mu} T{\mspace{11mu}\;}{cells}}{\%{\mspace{11mu}\;}{viable}\mspace{14mu}{control}\mspace{14mu}{cells}\mspace{14mu}{upon}\mspace{11mu}{coculture}\mspace{14mu}{with}\mspace{14mu}{modified}\mspace{14mu} T\mspace{14mu}{cells}}}{\frac{\%\mspace{14mu}{viable}\mspace{14mu}{target}{\mspace{11mu}\;}{cells}\mspace{14mu}{upon}\mspace{11mu}{coculture}\mspace{14mu}{with}\mspace{14mu}{non}\mspace{14mu}{modified}\mspace{14mu} T{\mspace{11mu}\;}{cells}}{\%\mspace{14mu}{viable}\mspace{14mu}{control}{\mspace{11mu}\;}{cells}\mspace{14mu}{upon}\mspace{11mu}{coculture}\mspace{14mu}{with}\mspace{14mu}{non}\mspace{14mu}{modified}\mspace{14mu} T{\mspace{11mu}\;}{cells}}}}}\mspace{14mu}$

With Suspension Target Cells:

ROR1-positive and ROR1-negative cells were respectively labeled with CellTrace CFSE and CellTrace Violet. About 2×10⁴ ROR1-positive cells were co-cultured with 2×10⁴ ROR1negative cells with 4×10⁵T cells in 0.1 ml per well in a 96-well plate. After a 4 hours incubation, the cells were harvested and stained with a fixable viability dye (eBioscience) and analyzed using the MACSQuant flow cytometer (Miltenyi).

The percentage of specific lysis was calculated using the previous formula.

EXAMPLES

Examples of CAR Polypeptide Sequences:

Hereafter, the sequences SEQ ID NO. 79 to 138 (with peptide signal) of 60 ROR1 scCARs are presented. These have CAR architecture of V1 to V6, and scFvs from 8 different antibodies: 2A2, 4A5, D10, G6, G3, H10, 2A4 and 1C11.

The versions V1, V3 and V5 (scCARs with the CD8α transmembrane domain) were assayed in the following experiments excepted for the scCAR with the 2A2 scFvs, for which only the V1 version was tested (as benchmark for comparison), this one having the same structure as described by Hudecek et al (2013).

Framed sequences correspond to preferred VH and VL sequences. However, VH and VL may be swapped to improve CAR efficiency according to the invention.

2A2 v1 (SEQ ID NO. 1 + SEQ ID NO. 79)

KLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR GRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 2A2 v2 (SEQ ID NO. 1 + SEQ ID NO. 80)

KKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKR RGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP R 2A2 v3 (SEQ ID NO. 1 + SEQ ID NO. 81)

CDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAP AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLYQGLSTATKDTYDALHMQALPPR 2A2 v4 (SEQ ID NO. 1 + SEQ ID NO. 82)

CDIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADA PAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRG KGHDGLYQGLSTATKDTYDALHMQALPPR 2A2 v5 (SEQ ID NO. 1 + SEQ ID NO. 83)

TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRP VQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 2A2 v6 (SEQ ID NO. 1 + SEQ ID NO. 84)

TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMR PVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 4A5 v1 (SEQ ID NO. 1 + SEQ ID NO. 85)

LLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRG RDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 4A5 v2 (SEQ ID NO. 1 + SEQ ID NO. 86)

KLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR GRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 4A5 v3 (SEQ ID NO. 1 + SEQ ID NO. 87)

DIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPA YQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG HDGLYQGLSTATKDTYDALHMQALPPR 4A5 v4 (SEQ ID NO. 1 + SEQ ID NO. 88)

DIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAP AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLYQGLSTATKDTYDALHMQALPPR 4A5 v5 (SEQ ID NO. 1 + SEQ ID NO. 89)

CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPV QTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 4A5 v6 (SEQ ID NO. 1 + SEQ ID NO. 90)

CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRP VQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR D10 v1 (SEQ ID NO. 1 + SEQ ID NO. 91)

QPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPE MGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR D10 v2 (SEQ ID NO. 1 + SEQ ID NO. 92)

KQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPE MGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR D10 v3 (SEQ ID NO. 1 + SEQ ID NO. 93)

APLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQG QNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGL YQGLSTATKDTYDALHMQALPPR D10 v4 (SEQ ID NO. 1 + SEQ ID NO. 94)

LALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQ GQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDG LYQGLSTATKDTYDALHMQALPPR D10 v5 (SEQ ID NO. 1 + SEQ ID NO. 95)

VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEE DGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEG LYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR D10 v6 (SEQ ID NO. 1 + SEQ ID NO. 96)

VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGKIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQE EDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Humanized D10-V1 (SEQ ID NO. 1 + SEQ ID NO. 97)

WAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQ GQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDG LYQGLSTATKDTYDALHMQALPPR Humanized D10-V2 (SEQ ID NO. 1 + SEQ ID NO. 98)

FFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQ QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD GLYQGLSTATKDTYDALHMQALPPR Humanized D10-V3 (SEQ ID NO. 1 + SEQ ID NO. 99)

PLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDG CSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYN ELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Humanized D10-V4 (SEQ ID NO. 1 + SEQ ID NO. 100)

PLSLRPEACRPAAGGAVHTRGLDFACDIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEED GCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLY NELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Humanized D10-V5 (SEQ ID NO. 1 + SEQ ID NO. 101

PVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLL SLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLG RREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY DALHMQALPPR Humanized D10-V6 (SEQ ID NO. 1 + SEQ ID NO. 102)

PVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIISFFLALTSTALLFLLF FLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNL GRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR G6 v1 (SEQ ID NO. 1 + SEQ ID NO. 103)

KKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKR RGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP R G6 v2 (SEQ ID NO. 1 + SEQ ID NO. 104)

RKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDK RRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP PR G6 v3 (SEQ ID NO. 1 + SEQ ID NO. 105)

FACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSAD APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRR GKGHDGLYQGLSTATKDTYDALHMQALPPR G6 v4 (SEQ ID NO. 1 + SEQ ID NO. 106)

FACDIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSA DAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERR RGKGHDGLYQGLSTATKDTYDALHMQALPPR G6 v5 (SEQ ID NO. 1 + SEQ ID NO. 107)

VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKOPFMR PVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR G6 v6 (SEQ ID NO. 1 + SEQ ID NO. 108)

VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFM RPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR G3 v1 (SEQ ID NO. 1 + SEQ ID NO. 109)

LLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRG RDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR G3 v2 (SEQ ID NO. 1 + SEQ ID NO. 110)

KLLYIFKOPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR GRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR G3 v3 (SEQ ID NO. 1 + SEQ ID NO. 111)

DIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPA YQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG HDGLYQGLSTATKDTYDALHMQALPPR G3 v4 (SEQ ID NO. 1 + SEQ ID NO. 112)

DIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAP AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLYQGLSTATKDTYDALHMQALPPR G3 v5 (SEQ ID NO. 1 + SEQ ID NO. 113)

CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPV QTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR G3 v6 (SEQ ID NO. 1 + SEQ ID NO. 114)

CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRP VQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR H10 v1 (SEQ ID NO. 1 + SEQ ID NO. 115)

KQPFMRPVQTTQEEDGCSCRFPFEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPE MGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR H10 v2 (SEQ ID NO. 1 + SEQ ID NO. 116)

FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP EMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR H10 v3 (SEQ ID NO. 1 + SEQ ID NO. 117)

WAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQ GQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDG LYQGLSTATKDTYDALHMQALPPR H10 v4 (SEQ ID NO. 1 + SEQ ID NO. 118)

FLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQ QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD GLYQGLSTATKDTYDALHMQALPPR H10 v5 (SEQ ID NO.1 + SEQ ID NO.119)

DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQE EDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR H10 v6 (SEQ ID NO. 1 + SEQ ID NO. 120)

DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGKIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQ EEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Humanized H10-v1 (SEQ ID NO. 1 + SEQ ID NO. 121)

VLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNEL NLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPR Humanized H10-v2 (SEQ ID NO. 1 + SEQ ID NO. 122)

FLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYN ELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA TKDTYDALHMQALPPR Humanized H10-v3 (SEQ ID NO. 1 + SEQ ID NO. 123)

PAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE GGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMA EAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Humanized H10-v4 (SEQ ID NO. 1 + SEQ ID NO. 124)

PAAGGAVHTRGLDFACDIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKOPFMRPVQTTQEEDGCSCRFPEEE EGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Humanized H10-v5 (SEQ ID NO. 1 + SEQ ID NO. 125)

PPKPKDILMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKR GRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR Humanized H10-v6 (SEQ ID NO. 1 + SEQ ID NO. 126)

PPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIISFFLALTSTALLFLLFFLTLRFSVVK RGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQA LPPR 2A4 v1 (SEQ ID NO. 1 + SEQ ID NO. 127)

IFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRD PEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 2A4 v2 (SEQ ID NO. 1 + SEQ ID NO. 128)

LYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR DPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 2A4 v3 (SEQ ID NO. 1 + SEQ ID NO. 129)

YIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAY QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPR 2A4 v4 (SEQ ID NO. 1 + SEQ ID NO. 130)

SFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAY QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPR 2A4 v5 (SEQ ID NO. 1 + SEQ ID NO. 131)

VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQT TQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 2A4 v6 (SEQ ID NO. 1 + SEQ ID NO. 132)

VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQ TTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKN PQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 1C11 v1 (SEQ ID NO. 1 + SEQ ID NO. 133)

IFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRD PEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 1C11 v2 (SEQ ID NO. 1 + SEQ ID NO. 134)

YIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR DPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 1C11 v3 (SEQ ID NO. 1 + SEQ ID NO. 135)

IWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQ QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD GLYQGLSTATKDTYDALHMQALPPR 1C11 v4 (SEQ ID NO. 1 + SEQ ID NO. 136)

SFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAY QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPR 1C11 v5 (SEQ ID NO. 1 + SEQ ID NO. 137)

VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQT TQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 1C11 v6 (SEQ ID NO. 1 + SEQ ID NO. 138)

VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQ TTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKN PQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

Example 1: Selection of ROR1-Positive and -Negative Cell Lines

To identify cell lines expressing different cell surface expression levels of ROR1, 12 human cell lines were analysed by flow cytometry using the Qifikit (Dako) and the anti-human ROR1 mAb clone 2A2 (see materials and methods). Nine of these cell lines had previously been described in the literature to be positive for ROR1 at the mRNA or protein level (Table 10).

TABLE 10 Cell lines reported to be positive for ROR1 in the literature cell line description cell type MDA-MB-231 adherent breast adenocarcinoma PC-3 adherent prostatic adenocarcinoma MDA-MB-468 adherent breast adenocarcinoma Hs746T adherent gastric carcinoma NCI-H1993 adherent non-small cell lung cancer HT-29 adherent colorectal adenocarcinoma A549 adherent human lung adenocarcinoma Cal51 adherent breast carcinoma Jeko-1 suspension Mantle cell lymphoma

Flow cytometry results indicate that MDA-MB-231 (ATCC® HTB-26), MDA-MB-468 (ATCC® HTB. 132), Hs746T (ATCC® HTB-135) and HT-29 (ATCC HTB-38) cells expressed the highest levels of ROR1 cell surface expression. Lower levels of ROR1 cell surface expression, approximately half that of the high expressing cell lines, were detected on PC-3 (ATCC.®. CRL-1435), NCI-H1993 (ATCC CRL 5909), A549 (ATCC CCL-185) and Jeko-1 (ATCC CRL-3006) cells (FIG. 5).

From these results, it is also observed that MCF-7, K562 and T cells did not express ROR1 and that the Cal51 expressed only very low levels of ROR1 on their surface.

From this cell lines, three adherent ones were selected to screen the activity of anti-ROR1 scCARs: PC-3 and MDA-MB-231 cells that express different levels of ROR1, and MCF-7 cells that are ROR1 negative.

Also, two suspension cell lines were selected to screen the activity of anti-ROR1 scCARs: Jeko-1 cells that are ROR1 positive and K562 cells that are ROR1 negative.

Example 2: Generation of Anti-ROR1 scCARs

A set of 18 ROR1-specific scCARs of 2nd generation (FIG. 1C) were created and tested in the following experiments.

They result from the fusion of the following building blocks of Tables 1-2:

-   -   scFvs from murine origin or humanized form from D10, G6, G3,         H10, 2A4 or 1C11;     -   one spacer: human FcγRIIIα, CD8α or IgG1 hinge;     -   the transmembrane domain of human CD8α and the costimulatory         domain of human 41BB;     -   the activation domain of human CD3

Different scFv were used in the scCARs to generate receptors of different binding affinities and different epitope specificities.

The mAbs D10 and G6 target the 3′ Ig like region and linker region between the Ig like domain and the CRD domain of ROR1 (FIG. 2)

The mAbs G3, H10 and 2A4 target the IgG like region of ROR1 (FIG. 2)

The mAb 1C11 targets the CRD domain of ROR1 (FIG. 2).

Three spacers of different length (16 AA, 45 AA and 231 AA) were used in the scCARs to try to optimize scCAR engagement by both proximal and distal epitopes of ROR1 (Guest et al., 2005).

Example 3: In Vitro Testing of Anti-ROR1 scCARs

The anti-ROR1 scCARs designed as previously were tested in human primary T cells using a two-step screening method presented in FIG. 6.

In the first step of the screening process, primary human T cells previously activated for 4-5 days with anti-CD28/CD3 beads and IL-2 were electroporated with mRNA encoding the 18 anti-ROR1 scCARs presented earlier. One day post electroporation, scCAR expression was assessed by flow cytometry and western blot, and scCAR-modified T cell activity was assessed by measuring T cells degranulation. The scCARs that were detected by western blot and that induced significant specific degranulation of T cells (≥20%) upon coculture with at least one ROR1 positive cell line were selected to pass through the second step of the screening process.

In the second step of the screening process, primary human T cells previously activated for 11-12 days with anti-CD28/CD3 beads and IL-2 were electroporated with mRNA encoding the anti-ROR1 scCARs selected after the first screening step. One-two days post electroporation, scCARs expression was assessed by flow cytometry, and scCAR-modified T cell activity was assessed by measuring effector functions of T cells (degranulation, IFNg production and cytotolytic activity. The scCARs that induced significant specific degranulation of T cells (≥20%), significant specific cytotoxicity of T cells (≥20% of target cell lysis) and significant production of IFNg by T cells (≥1500 pg/ml) upon co-culture with at least one ROR1 positive cell line were selected as potential scCAR candidates.

The above scCARs were systematically compared to a benchmark scCAR containing the 2A2 scFv fused to a short hinge (SEQ ID NO. 79) as this scCAR was previously shown to be functional in Hudecek et al., 2013.

a) Primary Screening of scCARs

scCAR Expression

First, the total expression of anti-ROR1 scCARs was assessed in T cells by western blot using an anti-human CD3 zeta mAb. It was observed that the scCARs that contained the intermediate (CD8α hinge) or the long spacer (IgG1 hinge) were strongly detected in T cells lysates whatever the scFv they harbored. However, for the scCARs that contained the short spacer (FcγRIIIα), those with the G6, G3 and H10 scFvs were well detected, whereas those with the D10, 2A4 and 1C11 scFvs were weakly or not detected (FIGS. 7A and 7B).

Then, the surface expression of anti-ROR1 scCARs on T cell was assessed by flow cytometry using anti-Fab or protein L (FIGS. 8A and 8B).

It was observed that:

-   -   the scCARs D10-v3, D10-v5, G6-v3, G6-v5, G3-v3, G3-v5, H10-v3,         H10-v5, 2A4-v3 and 2A4-v5 that were well detected in T cells         lysate by western blot were also well detected on the T cell         surface by flow cytometry;     -   the scCARs D10-v1, 2A4-v1 and 1C11-v1 that were not or almost         not detected in T cells lysate by western blot were also not         detected on the T cell surface by flow cytometry.

scCAR Activity

To assess scCAR activity, the degranulation of scCAR-modified T cells was analysed upon co-culture with ROR1-positive (MDA-MB-231 and PC-3) or ROR1-negative (MCF-7) cells.

It was observed that the T cells modified with the scCARs D10-v1, D10-v3, D10-v5, H10-v1, H10-v3 and H10-v5 degranulated significantly more (≥20%) upon co-culture with MDA-MB-231 and PC-3 cells than upon co-culture with MCF-7 cells (FIGS. 9A and 9B).

FIG. 9 A and FIG. 9B show that the non-modified T cells as well as the T cells modified with the scCARs H10 and D10 degranulate at all upon coculture with MDA-MB-231, PC-3 and MCF-7 cells much better than with the scCAR G6, G3, 2A4 and 1C11.

Conclusion of Primary Screening:

Altogether these results demonstrated that among the 18 anti-ROR1 scCARs designed within the present invention, 6 (D10-v1, D10-v3, D10-v5, H10-v1, H10-v3 and H10-v5) met the criteria to be selected to pass through the second step of the screening process.

b) Secondary Screening of scCARs

scCAR Expression

The surface expression of anti-ROR1 scCARs on T cells was assessed by flow cytometry using protein L.

It was observed that the scCARs D10-v3, D10-v5, H10-v3 and H10-v5 that were well detected on T cells electroporated 4-5 days post activation (FIGS. 8A and 8B) were not detected or only weakly detected on T cells electroporated 11-12 days post activation (FIG. 10).

scCAR Activity

First, to assess scCAR activity, the degranulation of scCAR-modified T cells was analysed upon co-culture with ROR1-positive (MDA-MB-231, PC-3 and Jeko-1) or ROR1-negative (MCF-7 and K562) cells.

It was observed that contrary to the T cells modified with the scCARs D10-v1, H10-v1 and H10-v5, the T cells modified with the scCARs D10-v3, D10-v5 and H10-v3 degranulated significantly more (≥20%) upon co-culture with MDA-MB-231 and PC-3 cells than upon co-culture with MCF-7 cells or in media alone (FIG. 11).

Then was analysed the production of IFNγ by scCAR-modified T cells upon co-culture with ROR1-positive (MDA-MB-231, PC-3 and Jeko-1) or ROR1-negative (MCF-7 and K562) cells.

It was observed that contrary to the T cells modified with the scCARs D10-v1, H10-v1 and H10-v5, the T cells modified with the scCARs D10-v3, D10-v5 and H10-v3 produced significantly more IFNγ (≥1500 pg/ml) upon co-culture with MDA-MB-231, PC-3 and Jeko-1 cells than upon co-culture with MCF-7, K562 cells or media alone (FIG. 12).

Finally, the cytotoxic activity of scCAR-modified T cells was analysed upon co-culture with ROR-1-negative (MCF-7 and K562) or ROR1-positive (MDA-MB-231, PC-3 and Jeko-1) cells.

It was observed that contrary to the T cells modified with the scCARs D10-v1, H10-v1 and H10-v5, the T cells modified with the scCARs D10-v3, D10-v5 and H10-v3 specifically killed more than 20% of PC-3 cells in co-culture (FIG. 13). Although the T cells modified with the scCARs D10-v3 and H10-v3 also killed more than 20% of MDA-MB-231 and Jeko-1 cells in co-culture, this was not the case for the T cells modified with the scCAR D10-v5 (FIG. 13 and FIG. 14).

Conclusion of Secondary Screening:

Altogether these results demonstrated that, from all the scCARs tested, D10-v3, D10-v5 and H10-v3 represent the most valuable scCARs.

A slight advantage for the 2 CARs of version V2 can be noted in terms of induction of cytotoxicity (better responses against the different ROR1-positive cell lines tested).

Example 4: Proliferation of TCRα Inactivated Cells Expressing a ROR1-CAR

FIG. 15 shows a schematic representation of TCRα inactivation by using rare cutting endonuclease. Heterodimeric TALE-nuclease targeting two 17-bp long sequences (called half targets) separated by an 15-bp spacer within T-cell receptor alpha constant chain region (TRAC) gene were designed and produced. Each half target is recognized by repeats of the half TALE-nucleases listed in Table 11.

TABLE 11 TAL-nucleases targeting TCRalpha gene Target Target sequence Repeat sequence Half TALE-nuclease TRAC_T01 TTGTCCCACAGATATCC Repeat TRAC_T01-L TRAC_T01-L TALEN Agaaccctgaccctg (SEQ ID NO: 140) (SEQ ID NO: 142) CCGTGTACCAGCTGAGA Repeat TRAC_T01-R TRAC_T01-R TALEN (SEQ ID NO: 139) (SEQ ID NO: 141) (SEQ ID NO: 143)

Each TALE-nuclease construct was subcloned using restriction enzyme digestion in a mammalian expression vector under the control of the T7 promoter. mRNA encoding TALE-nuclease cleaving TRAC genomic sequence were synthesized from plasmid carrying the coding sequence downstream from the T7 promoter.

Purified T cells preactivated during 72 hours with anti-CD3/CD28 coated beads were transfected with each of the 2 mRNAs encoding both half TRAC T01 TALE-nucleases. 48 hours post-transfection, different groups of T cells from the same donor were respectively transduced with a lentiviral vector encoding one of the ROR1 CAR previously described (SEQ ID NO: 79 to 138). 2 days post-transduction, CD3_(NEG) cells were purified using anti-CD3 magnetic beads and 5 days post-transduction cells were reactivated with soluble anti-CD28 (5 μg/ml).

Cell proliferation was followed for up to 30 days after reactivation by counting cell 2 times per week. Increased proliferation in TCR alpha inactivated cells expressing the ROR1 CARs, especially when reactivated with anti-CD28, was observed compared to non-transduced cells.

To investigate whether the human T cells expressing the ROR1 CAR display activated state, the expression of the activation marker CD25 are analyzed by FACS 7 days post transduction. The purified cells transduced with the lentiviral vector encoding ROR1 CAR assayed for CD25 expression at their surface in order to assess their activation in comparison with the non-transduced cells. Increased CD25 expression was indicative of anti-CD28 reactivation or no reactivation conditions.

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The invention claimed is:
 1. A ROR1 (NTRKR1) specific chimeric antigen receptor (CAR) comprising: (a) an extracellular ligand binding-domain comprising a variable heavy chain comprising CDR regions as set forth in SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:56, and a variable light chain comprising CDR regions as set forth in SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO:61, (b) a hinge comprising an amino acid sequence at least 95 percent identical to SEQ ID NO:4, (c) a transmembrane domain comprising an amino acid sequence at least 95 percent identical to SEQ ID NO:6, and (d) a cytoplasmic domain comprising (i) a signaling domain comprising an amino acid sequence at least 95 percent identical to SEQ ID NO:9 and (ii) a co-stimulatory domain comprising an amino acid sequence at least 95 percent identical to SEQ ID NO:8.
 2. The ROR1 specific CAR according to claim 1, wherein the transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:6.
 3. The ROR1 specific CAR according to claim 1, wherein the hinge comprises the amino acid sequence set forth in SEQ ID NO:4.
 4. The ROR1 specific CAR according to claim 1, wherein said extracellular ligand binding-domain comprises VH and VL chains with at least 95 percent sequence identity to SEQ ID NO:53 and SEQ ID NO:58, respectively.
 5. The ROR1 specific CAR according to claim 1, wherein said extracellular ligand binding-domain comprises humanized V_(H) and V_(L) chains from an H10 antibody.
 6. The ROR1 specific CAR according to claim 5, wherein said humanized V_(H) chain comprises the amino acid sequence encoded by SEQ ID NO:57, and said humanized V_(L) chain comprises the amino acid sequence encoded by SEQ ID NO:62.
 7. The ROR1 specific CAR according to claim 1, wherein said CAR comprises an amino acid sequence at least 95 percent identical to the sequence set forth in SEQ ID NO:117.
 8. The ROR1 specific CAR according to claim 1, wherein said co-stimulatory domain comprises the amino acid sequence set forth in SEQ ID NO:8.
 9. The ROR1 specific CAR according to claim 1, wherein said signaling domain comprises the amino acid sequence set forth in SEQ ID NO:9.
 10. The ROR1 specific CAR according to claim 1, further comprising a signal peptide. 